• Degen M, Barron P, Natarajan E, Widlund HR, Rheinwald JG (2013) RSK activation of translation factor eIF4B drives abnormal increases of Laminin γ2 and MYC protein during neoplastic progression to squamous cell carcinoma.  PLoS One 10: e78979.  Abstract  |  PDF
  • Pinto AP, Degen M, Barron P, Crum CP, Rheinwald JG (2013) Phosphorylated S6 as an immunohistochemical biomarker of vulvar intraepithelial neoplasia. Mod Pathol 26: 1498–1507.  Abstract  |  PDF
  • Rheinwald JG (2013) The quest to derive keratinocytes from pluripotent stem cells. Current Pathobiol Rep 1: 119-128.  Abstract  |  PDF
  • Degen  M, Natarajan E, Barron P, Widlund HR, Rheinwald JG (2012) MAPK/ERK-dependent translation factor hyperactivation and dysregulated Laminin γ2 expression in oral dysplasia and squamous cell carcinoma.  Am J Path 180: 2462-2478. Abstract | PDF
  • Wei W, Barron PD, Rheinwald JG (2010) Modulation of TGFβ-inducible hypermotility by EGF and other factors in human prostate epithelial cells and keratinocytes. In Vitro Cell Dev Biol Anim 46: 841-855. Abstract | PDF
  • Zhang W, Choi J, Zeng W, Rogers SA, Alyea EP, Rheinwald JG, Canning CM, Brusic V, Sasada T, Reinherz EL, Ritz J, Soiffer RJ, Wu CJ (2010) Graft-versus-leukemia antigen CML66 elicits coordinated B-cell and T-cell immunity after donor lymphocyte infusion. Clin Cancer Res 16: 2729-2739. PDF | Abstract
  • Utikal J, Polo JM, Stadtfeld M, Maherali N, Kulalert W, Walsh RM, Khalil A, Rheinwald JG, Hochedlinger K. (2009) Immortalization eliminates a roadblock during cellular reprogramming into iPS cells. Nature 460:1145-1148. Abstract | PDF
  • Dabelsteen S, Hercule P, Barron P, Rice M, Dorsainville G, Rheinwald JG (2009) Epithelial cells derived from human embryonic stem cells display p16(INK4A) senescence, hypermotility, and differentiation properties shared by many p63+ somatic cell types. Stem Cells 27: 1388-1399. Abstract | PDF
  • Natarajan N, Omobono JD II, Guo Z, Hopkinson S, Lazar AJF, Brenn T, Jones JC, Rheinwald JG (2006) A keratinocyte hypermotility/growth arrest response involving Laminin 5 and p16INK4A activated in wound healing and senescence. Am J Pathol 168: 1821-1837. Abstract | PDF
  • Iuchi S, Dabelsteen S, Easley K, Rheinwald JG, Green H (2006). Immortalized keratinocyte lines derived from human embryonic stem cells. Proc Natl Acad Sci USA. Abstract | PDF
  • Natarajan E, Omobono JD, 2nd, Jones JC, Rheinwald JG (2005). Co-expression of p16INK4A and laminin 5 by keratinocytes: a wound-healing response coupling hypermotility with growth arrest that goes awry during epithelial neoplastic progression. J Investig Dermatol Symp Proc 10: 72-85. Abstract | PDF
  • Gordon GJ, Rockwell GN, Jensen RV, Rheinwald JG, Glickman JN, Aronson JP, Pottorf BJ, Nitz MD, Richards WG, Sugarbaker DJ, Bueno R (2005). Identification of novel candidate oncogenes and tumor suppressors in malignant pleural mesothelioma using large-scale transcriptional profiling. Am J Pathol 166: 1827-1840. Abstract | PDF
  • Kim M, Yang H, Kim SK, Reche PA, Tirabassi RS, Hussey RE, Chishti Y, Rheinwald JG, Morehead TJ, Zech T, Damon IK, Welsh RM, Reinherz EL (2004). Biochemical and functional analysis of smallpox growth factor (SPGF) and anti-SPGF monoclonal antibodies. J Biol Chem 279: 25838-25848. Abstract | PDF
  • Natarajan E, Saeb M, Crum CP, Woo SB, McKee PH, Rheinwald JG (2003) Co-expression of p16(INK4A) and laminin 5 gamma2 by microinvasive and superficial squamous cell carcinomas in vivo and by migrating wound and senescent keratinocytes in culture. Am J Pathol 163: 477-491. Abstract | PDF
  • Kleine-Lowinski K, Rheinwald JG, Fichorova RN, Anderson DJ, Basile J, Munger K, Daly CM, Rosl F, Rollins BJ (2003) Selective suppression of monocyte chemoattractant protein-1 expression by human papillomavirus E6 and E7 oncoproteins in human cervical epithelial and epidermal cells. Int J Cancer 107: 407-415. Abstract | PDF
  • Rheinwald JG (2003) Becoming a scientist: what I learned from Gunny. Biochem Biophys Res Commun 312: 61-64. PDF
  • Rheinwald JG, Hahn WC, Ramsey MR, Wu JY, Guo Z, Tsao H, De Luca M, Catricala C, O'Toole KM (2002) A two-stage, p16(INK4A)- and p53-dependent keratinocyte senescence mechanism that limits replicative potential independent of telomere status. Mol Cell Biol 22: 5157-5172. Abstract | PDF
  • Verselis SJ, Rheinwald JG, Fraumeni JF, Jr., Li FP (2000) Novel p53 splice site mutations in three families with Li-Fraumeni syndrome. Oncogene 19: 4230-4235. Abstract | PDF
  • Dickson MA, Hahn WC, Ino Y, Ronfard V, Wu JY, Weinberg RA, Louis DN, Li FP, Rheinwald JG (2000) Human keratinocytes that express hTERT and also bypass a p16(INK4a)- enforced mechanism that limits life span become immortal yet retain normal growth and differentiation characteristics. Mol Cell Biol 20: 1436-1447. Abstract | PDF
  • Schon M, Benwood J, O'Connell-Willstaedt T, Rheinwald JG (1999) Human sweat gland myoepithelial cells express a unique set of cytokeratins and reveal the potential for alternative epithelial and mesenchymal differentiation states in culture. J Cell Sci 112: 1925-1936. Abstract | PDF
  • Fichorova RN, Rheinwald JG, Anderson DJ (1997) Generation of papillomavirus-immortalized cell lines from normal human ectocervical, endocervical, and vaginal epithelium that maintain expression of tissue-specific differentiation proteins. Biol Reprod 57: 847-855. Abstract | PDF
  • Murphy JE, Rheinwald JG (1997) Intraperitoneal injection of genetically modified, human mesothelial cells for systemic gene therapy. Hum Gene Ther 8: 1867-1879. Abstract | PDF
  • Schrager HM, Rheinwald JG, Wessels MR (1996) Hyaluronic acid capsule and the role of streptococcal entry into keratinocytes in invasive skin infection. J Clin Invest 98: 1954-1958. Abstract | PDF
  • Schon M, Rheinwald JG (1996) A limited role for retinoic acid and retinoic acid receptors RAR alpha and RAR beta in regulating keratin 19 expression and keratinization in oral and epidermal keratinocytes. J Invest Dermatol 107: 428-438. Abstract | PDF
  • Park S, Schalling M, Bernard A, Maheswaran S, Shipley GC, Roberts D, Fletcher J, Shipman R, Rheinwald J, Demetri G, et al. (1993) The Wilms tumour gene WT1 is expressed in murine mesoderm-derived tissues and mutated in a human mesothelioma. Nat Genet 4: 415-420. Abstract | PDF
  • Lindberg K, Brown ME, Chaves HV, Kenyon KR, Rheinwald JG (1993) In vitro propagation of human ocular surface epithelial cells for transplantation. Invest Ophthalmol Vis Sci 34: 2672-2679. Abstract | PDF
  • Hu L, Crowe DL, Rheinwald JG, Chambon P, Gudas LJ (1991) Abnormal expression of retinoic acid receptors and keratin 19 by human oral and epidermal squamous cell carcinoma cell lines. Cancer Res 51: 3972-3981. Abstract | PDF
  • Crowe DL, Hu L, Gudas LJ, Rheinwald JG (1991) Variable expression of retinoic acid receptor (RAR beta) mRNA in human oral and epidermal keratinocytes; relation to keratin 19 expression and keratinization potential. Differentiation 48: 199-208. Abstract | PDF
  • Lindberg K, Rheinwald JG (1990) Three distinct keratinocyte subtypes identified in human oral epithelium by their patterns of keratin expression in culture and in xenografts. Differentiation 45: 230-241. Abstract | PDF
  • Henrard DR, Thornley AT, Brown ML, Rheinwald JG (1990) Specific effects of ras oncogene expression on the growth and histogenesis of human epidermal keratinocytes. Oncogene 5: 475-481. Abstract | PDF
  • Demetri GD, Ernst TJ, Pratt ES, Zenzie BW, Rheinwald JG, Griffin JD (1990) Expression of ras oncogenes in cultured human cells alters the transcriptional and posttranscriptional regulation of cytokine genes. J Clin Invest 86: 1261-1269. Abstract | PDF
  • Rollins BJ, O'Connell TM, Bennett G, Burton LE, Stiles CD, Rheinwald JG (1989) Environment-dependent growth inhibition of human epidermal keratinocytes by recombinant human transforming growth factor-beta. J Cell Physiol 139: 455-462. Abstract | PDF
  • Lindberg K, Rheinwald JG (1989) Suprabasal 40 kd keratin (K19) expression as an immunohistologic marker of premalignancy in oral epithelium. Am J Pathol 134: 89-98. Abstract | PDF
  • Demetri GD, Zenzie BW, Rheinwald JG, Griffin JD (1989) Expression of colony-stimulating factor genes by normal human mesothelial cells and human malignant mesothelioma cells lines in vitro. Blood 74: 940-946. Abstract | PDF
  • Cicila GT, O'Connell TM, Hahn WC, Rheinwald JG (1989) Cloned cDNA sequence for the human mesothelial protein 'mesosecrin' discloses its identity as a plasminogen activator inhibitor (PAI-1) and a recent evolutionary change in transcript processing. J Cell Sci 94: 1-10. Abstract | PDF
  • Tubo RA, Rheinwald JG (1987) Normal human mesothelial cells and fibroblasts transfected with the EJras oncogene become EGF-independent, but are not malignantly transformed. Oncogene Res 1: 407-421. Abstract | PDF
  • Rheinwald JG, Jorgensen JL, Hahn WC, Terpstra AJ, O'Connell TM, Plummer KK (1987) Mesosecrin: a secreted glycoprotein produced in abundance by human mesothelial, endothelial, and kidney epithelial cells in culture. J Cell Biol 104: 263-275. Abstract | PDF
  • Rheinwald JG, O'Connell TM (1985) Intermediate filament proteins as distinguishing markers of cell type and differentiated state in cultured human urinary tract epithelia. Ann N Y Acad Sci 455: 259-267. PDF
  • La Rocca PJ, Rheinwald JG (1984) Anchorage-independent growth of normal human mesothelial cells: a sensitive bioassay for EGF which discloses the absence of this factor in fetal calf serum. In Vitro Cell Dev Biol 21: 67-72. Abstract | PDF
  • Weichselbaum R, Dahlberg W, Little JB, Ervin TJ, Miller D, Hellman S, Rheinwald JG (1984) Cellular X-ray repair parameters of early passage squamous cell carcinoma lines derived from patients with known responses to radiotherapy. Br J Cancer 49: 595-601. Abstract | PDF
  • Nadakavukaren KK, Summerhayes IC, Salcedo BF, Rheinwald JG, Chen LB (1984) A monoclonal antibody recognizing a keratin filament protein in a subset of transitional and glandular epithelia. Differentiation 27: 209-220. Abstract | PDF
  • LaRocca PJ, Rheinwald JG (1983) Coexpression of simple epithelial keratins and vimentin by human mesothelium and mesothelioma in vivo and in culture. Cancer Res 44: 2991-2999. Abstract | PDF
  • Allen-Hoffmann BL, Rheinwald JG (1984) Polycyclic aromatic hydrocarbon mutagenesis of human epidermal keratinocytes in culture. Proc Natl Acad Sci U S A 81: 7802-7806. Abstract | PDF
  • Kim KH, Rheinwald JG, Fuchs EV (1983) Tissue specificity of epithelial keratins: differential expression of mRNAs from two multigene families. Mol Cell Biol 3: 495-502. Abstract | PDF
  • Connell ND, Rheinwald JG (1983) Regulation of the cytoskeleton in mesothelial cells: reversible loss of keratin and increase in vimentin during rapid growth in culture. Cell 34: 245-253. Abstract | PDF
  • Wu YJ, Parker LM, Binder NE, Beckett MA, Sinard JH, Griffiths CT, Rheinwald JG (1982) The mesothelial keratins: a new family of cytoskeletal proteins identified in cultured mesothelial cells and nonkeratinizing epithelia. Cell 31: 693-703. Abstract | PDF
  • Rheinwald JG (1982) Human squamous cell carcinoma in culture: a defect in terminal differentiation and its relation to malignancy. Natl Cancer Inst Monogr 60: 133-138. Abstract | PDF
  • Wu YJ, Rheinwald JG (1981) A new small (40 kd) keratin filament protein made by some cultured human squamous cell carcinomas. Cell 25: 627-635. Abstract | PDF
  • Rheinwald JG, Beckett MA (1981) Tumorigenic keratinocyte lines requiring anchorage and fibroblast support cultures from human squamous cell carcinomas. Cancer Res 41: 1657-1663. Abstract | PDF
  • Didinsky JB, Rheinwald JG (1981) Failure of hydrocortisone or growth factors to influence the senescence of fibroblasts in a new culture system for assessing replicative lifespan. J Cell Physiol 109: 171-179. Abstract | PDF
  • Rheinwald JG, Beckett MA (1980) Defective terminal differentiation in culture as a consistent and selectable character of malignant human keratinocytes. Cell 22: 629-632. Abstract | PDF
  • Rheinwald JG, Green H (1977) Epidermal growth factor and the multiplication of cultured human epidermal keratinocytes. Nature 265: 421-424. Abstract | PDF
  • Green H, Rheinwald JG, Sun TT (1977) Properties of an epithelial cell type in culture: the epidermal keratinocyte and its dependence on products of the fibroblast. Prog Clin Biol Res 17: 493-500. Abstract
  • Rheinwald JG, Green H (1975) Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6: 331-343. Abstract | PDF
  • Rheinwald JG, Green H (1975) Formation of a keratinizing epithelium in culture by a cloned cell line derived from a teratoma. Cell 6: 317-330. Abstract | PDF
  • Rheinwald JG, Green H (1974) Growth of cultured mammalian cells on secondary glucose sources. Cell 2: 287-293. Abstract | PDF
  • Rheinwald JG, Chakrabarty AM, Gunsalus IC (1973) A transmissible plasmid controlling camphor oxidation in Pseudomonas putida. Proc Natl Acad Sci USA 70: 885-889. Abstract | PDF

Degen M, Barron P, Natarajan E, Widlund HR, Rheinwald JG (2013) RSK activation of translation factor eIF4B drives abnormal increases of Laminin γ2 and MYC protein during neoplastic progression to squamous cell carcinoma.  PLoS One 10: e78979.

Department of Dermatology, Brigham and Women’s Hospital and Harvard Skin Disease Research Center, Boston, MA 02115, and Section of Oral and Maxillofacial Pathology, University of Connecticut Health Center, Farmington, CT 06030, USA

Overexpression of the basement membrane protein Laminin γ2 (Lamγ2) is a feature of many epidermal and oral dysplasias and all invasive squamous cell carcinomas (SCCs).  This abnormality has potential value as an immunohistochemical biomarker of premalignancy but its mechanism has remained unknown.  We recently reported that Lamγ2 overexpression in culture is the result of deregulated translation controls and depends on the MAPK-RSK signaling cascade.  Here we identify eIF4B as the RSK downstream effector responsible for elevated Lamγ2 as well as MYC protein in neoplastic epithelial cells.  Premalignant dysplastic keratinocytes, SCC cells, and keratinocytes expressing the HPV16 E6 oncoprotein all displayed MAPK-RSK and mTOR-S6K1 activation and overexpressed Lamγ2 and MYC in culture.  Immunohistochemical staining of oral dysplasias and SCCs for distinct, RSK- and S6K1-specific S6 phosphorylation events revealed that their respective upstream pathways become hyperactive at the same time during neoplastic progression.  However, pharmacologic kinase inhibitor studies in culture revealed that Lamγ2 and MYC overexpression depends on MAPK-RSK activity, independent of PI3K-mTOR-S6K1.  eIF4B knockdown reduced Lamγ2 and MYC protein expression, consistent with the known requirement for eIF4B to translate mRNAs with long, complex 5’ untranslated regions (5’-UTRs).  Accordingly, expression of a luciferase reporter construct preceded by the Lamγ2 5’-UTR proved to be RSK-dependent and mTOR-independent.  These results demonstrate that RSK activation of eIF4B is causally linked to elevated Lamγ2 and MYC protein levels during neoplastic progression to invasive SCC.  These findings have potential clinical significance for identifying premalignant lesions and for developing targeted drugs to treat SCC. PDF | Back


Pinto AP, Degen M, Barron P, Crum CP, Rheinwald JG (2013) Phosphorylated S6 as an immunohistochemical biomarker of vulvar intraepithelial neoplasia. Mod Pathol 26: 1498–1507.

Division of Women's and Perinatal Pathology, Department of Pathology, and Department of Dermatology, Brigham and Women's Hospital, and Harvard Skin Disease Research Center, Boston, MA, 02115 USA

As life expectancy lengthens, cases of non-viral-associated vulvar squamous cell carcinoma and its precursor lesion, so-called differentiated vulvar intraepithelial neoplasia (VIN), continue to increase in frequency.  Differentiated VIN often is difficult to recognize and failure to detect it prior to invasion results in morbidity and mortality.  Thus, identification of a reliable biomarker for this type of lesion would be of great clinical benefit.  Our recent studies have identified activation (ser235/236 phosphorylation) of ribosomal protein S6 (p-S6) in basal epithelial cells as an event that precedes and accompanies laminin γ2 overexpression in most preinvasive oral dysplasias.  To test this as a potential biomarker of vulvar dysplasia, we immunostained seven differentiated VINs and nine papillomavirus-related, “classic” VINs, most of which were associated with carcinoma, for p-S6.  All carcinomas, all differentiated VINs, and most classic VINs contained regions of p-S6 staining in the basal layer, whereas basal and parabasal cells of normal vulvar epithelium and hyperplastic and inflamed lesions lacking cellular atypia were p-S6 negative. Laminin γ2 was expressed in a subset of VINs, always occurring within basal p-S6 positive regions, as we had found previously for oral dysplasias.  Lichen sclerosus is considered a potential precursor of vulvar carcinoma.  Two lichen sclerosus lesions of patients with a concurrent carcinoma and one of six lichen sclerosus lesions without atypia or known concurrent carcinoma were basal p-S6 positive.  In summary, there is a distinct difference in p-S6 basal cell layer staining between benign and neoplastic vulvar squamous epithelium, with consistent staining of differentiated VIN and of some lichen sclerosus lesions.  These results support further studies to assess the potential of p-S6 as a biomarker to identify vulvar lesions at risk of progressing to invasive cancer. PDF | Back


Rheinwald JG (2013) The quest to derive keratinocytes from pluripotent stem cells. Current Pathobiol Rep 1: 119-128.

Department of Dermatology, Brigham and Women’s Hospital, Harvard Medical School, and the Harvard Skin Disease Research Center, Boston, MA 02115 USA

The derivation of keratinocytes from pluripotent stem cells (PSC) is a worthy goal.  Epidermal and corneal keratinocytes cultured from normal human tissue have been very well-characterized for their growth and differentiation properties and their ability to regenerate permanent tissue as autologous transplants.  PSC-derived keratinocytes would therefore be ideal for establishing proof-of-principle that normal, well-defined, and functional somatic cell types can be derived from PSC cells and used successfully in tissue replacement therapies.  Much progress has been made to identify experimental protocols for deriving cells that display some keratinocyte properties from ES and iPS cells.  However, the proliferative potential of these cells typically is very limited and many features of these cells are common to all members of the large family of p63 epithelial cell types.  Thus many lines of investigation remain to be examined more thoroughly in this important and exciting field. PDF | Back


MAPK/ERK-dependent translation factor hyperactivation and dysregulated Laminin γ2 expression in oral dysplasia and squamous cell carcinoma.

Degen  M, Natarajan E, Barron P, Widlund HR, Rheinwald JG (2012)  Am J Path 180: 2462-2478.

Department of Dermatology,* Brigham and Women’s Hospital and Harvard Skin Disease Research Center, Boston, MA; and the Section of Oral and Maxillofacial Pathology, University of Connecticut Health Center, Farmington, CT

Lesions displaying a variety of dysplastic changes precede invasive oral and epidermal squamous cell carcinoma (SCC); however, there are no histopathologic criteria for either confirming or staging premalignancy.  SCCs and dysplasias frequently contain cells that abnormally express the γ2 subunit of laminin-332.  We developed cell culture models to investigate γ2 dysregulation.  Normal human keratinocytes displayed density-dependent repression of γ2, whereas premalignant keratinocytes and SCC cells overexpressed γ2 and secreted laminin assembly intermediates.  Neoplastic cells had hyperactive EGFR/MAPK(ERK) signaling coordinate with overexpressed γ2, and EGFR and MEK inhibitors normalized γ2 expression.  Keratinocytes engineered to express HPV16 E6 or activated mutant HRAS, cRAF1, or MEK1 lost density repression of γ2 and shared with neoplastic cells signaling abnormalities downstream of ERK, including increased phosphorylation of S6 and eIF4 translation factors.  Notably, qPCR results revealed that γ2 overexpression was not accompanied by increased γ2 mRNA levels, consistent with ERK-dependent, eIF4B-mediated translation initiation of the stem-looped, 5’-untranslated region of γ2 mRNA in neoplastic cells.  Inhibitors of MEK, but not of TORC1/2, blocked S6 and eIF4B phosphorylation and γ2 overexpression.  Immunostaining of oral dysplasias identified γ2 overexpression occurring within fields of basal cells that had elevated p-S6 levels.  These results reveal a causal relationship between ERK-dependent translation factor activation and laminin γ2 dysregulation and identify new markers of preinvasive neoplastic change during progression to SCC. PDF | Back

Modulation of TGFβ-inducible hypermotility by EGF and other factors in human prostate epithelial cells and keratinocytes.

Wei W, Barron PD, Rheinwald JG In Vitro Cell Dev Biol Anim (2010) 46: 841-855.

Department of Dermatology and Harvard Skin Disease Research Center, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115

Keratinocytes migrating from a wound edge or initiating malignant invasion greatly increase their expression of the basement membrane protein Laminin-322 (Lam332).  In culture, keratinocytes initiate sustained directional hypermotility when plated onto an incompletely processed form of Lam332 (Lam332’) or when treated with TGFβ, an inducer of Lam332 expression.  The development and tissue architecture of stratified squamous and prostate epithelia are very different, yet the basal cells of both express p63, α6β4 integrin, and Lam332.  Keratinocytes and prostate epithelial cells grow well in nutritionally-optimized culture media with pituitary extract and certain mitogens.  We report that prostate epithelial cells display hypermotility responses indistinguishable from those of keratinocytes.  Several culture medium variables attenuated TGFβ-induced hypermotility, including Ca++, serum, and some pituitary extract preparations, without impairing growth, TGFβ growth-inhibition, or hypermotility on Lam322’.  Distinct from its role as a mitogen, EGF proved to be a required cofactor for TGFβ-induced hypermotility and could not be replaced by HGF or KGF.  Prostate epithelial cells have a short replicative lifespan, restricted both by p16INK4A and telomere-related mechanisms.  We immortalized the normal prostate epithelial cell line HPrE-1 by transduction to express bmi1 and TERT.  Prostate epithelial cells lose expression of p63, β4 integrin, and Lam332 when they transform to invasive carcinoma.  In contrast, HPrE-1/bmi1/TERT cells retained expression of these proteins and normal TGFβ signaling and hypermotility for >100 doublings.  Thus, keratinocytes and prostate epithelial cells possess common hypermotility and senescence mechanisms and immortalized prostate cell lines can be engineered using defined methods to yield cells retaining normal properties. PDF | Back


Graft-versus-leukemia antigen CML66 elicits coordinated B-cell and T-cell immunity after donor lymphocyte infusion.

Zhang W, Choi J, Zeng W, Rogers SA, Alyea EP, Rheinwald JG, Canning CM, Brusic V, Sasada T, Reinherz EL, Ritz J, Soiffer RJ, Wu CJ (2010) Clin Cancer Res 16: 2729-2739.

PURPOSE: The target antigens of graft-versus-leukemia that are tumor associated are incompletely characterized. EXPERIMENTAL DESIGN: We examined responses developing against CML66, an immunogenic antigen preferentially expressed in myeloid progenitor cells identified from a patient with chronic myelogenous leukemia who attained long-lived remission following CD4+ donor lymphocyte infusion (DLI). RESULTS: From this patient, CML66-reactive CD8+ T-cell clones were detected against an endogenously presented HLA-B*4403-restricted epitope (HDVDALLW). Neither CML66-specific antibody nor T-cell responses were detectable in peripheral blood before DLI. However, by 1 month after DLI, CD8+ T cells were present in peripheral blood and at 10-fold higher frequency in marrow. Subsequently, plasma antibody to CML66 developed in association with disease remission. Donor-derived CML66-reactive T cells were detected at low levels in vivo in marrow before DLI by ELISpot and by a nested PCR-based assay to detect clonotypic T-cell receptor sequences but not in blood of the patient pre-DLI nor of the graft donor. CONCLUSIONS: CD4+ DLI results in rapid expansion of preexisting marrow-resident leukemia-specific donor CD8+ T cells, followed by a cascade of antigen-specific immune responses detectable in blood. Our single-antigen analysis thus shows that durable posttransplant tumor immunity is directed in part against nonpolymorphic overexpressed leukemia antigens that elicit coordinated cellular and humoral immunity. PDF | Back


Immortalization eliminates a roadblock during cellular reprogramming into iPS cells.

Utikal J, Polo JM, Stadtfeld M, Maherali N, Kulalert W, Walsh RM, Khalil A, Rheinwald JG, Hochedlinger K.

Massachusetts General Hospital Cancer Center and Center for Regenerative Medicine, Harvard Stem Cell Institute, 185 Cambridge Street, Boston, Massachusetts 02114, USA.

The overexpression of defined transcription factors in somatic cells results in their reprogramming into induced pluripotent stem (iPS) cells. The extremely low efficiency and slow kinetics of in vitro reprogramming suggest that further rare events are required to generate iPS cells. The nature and identity of these events, however, remain elusive. We noticed that the reprogramming potential of primary murine fibroblasts into iPS cells decreases after serial passaging and the concomitant onset of senescence. Consistent with the notion that loss of replicative potential provides a barrier for reprogramming, here we show that cells with low endogenous p19(Arf) (encoded by the Ink4a/Arf locus, also known as Cdkn2a locus) protein levels and immortal fibroblasts deficient in components of the Arf-Trp53 pathway yield iPS cell colonies with up to threefold faster kinetics and at a significantly higher efficiency than wild-type cells, endowing almost every somatic cell with the potential to form iPS cells. Notably, the acute genetic ablation of Trp53 (also known as p53) in cellular subpopulations that normally fail to reprogram rescues their ability to produce iPS cells. Our results show that the acquisition of immortality is a crucial and rate-limiting step towards the establishment of a pluripotent state in somatic cells and underscore the similarities between induced pluripotency and tumorigenesis. PDF | Back


Epithelial Cells Derived from Human Embryonic Stem Cells Display P16(INK4A) Senescence, Hypermotility, and Differentiation Properties Shared by Many P63(+) Somatic Cell Types.

Dabelsteen S, Hercule P, Barron P, Rice M, Dorsainville G, Rheinwald JG.

Department of Dermatology and Harvard Skin Disease Research Center, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA.

Human embryonic stem (hES) cells can generate cells expressing p63, K14, and involucrin, which have been proposed to be keratinocytes.  These hES-derived, keratinocyte-like (hESderK) cells form epithelioid colonies when cultured in a fibroblast feeder system optimal for normal tissue-derived keratinocytes, but they have a very short replicative lifespan unless engineered to express HPV16 E6E7.  We report here that hESderK cells undergo senescence associated with p16INK4A expression, unrelated to telomere status.  Transduction to express bmi1, a repressor of the p16INK4A/p14ARF locus, conferred upon hESderK cells and keratinocytes a substantially extended lifespan.  When exposed to TGFb or to an incompletely processed form of Laminin-332, three lifespan-extended or immortalized hESderK lines we studied became directionally hypermotile, a wound healing and invasion response previously characterized in keratinocytes.  In organotypic culture, hESderK cells stratified and expressed involucrin and K10, as do epidermal keratinocytes in vivo.  However, their growth requirements were less stringent than keratinocytes.  We then extended the comparison to endoderm-derived, p63+/K14+ urothelial and tracheobronchial epithelial cells.  Primary and immortalized lines of these cell types had growth requirements and hypermotility responses similar to keratinocytes and bmi1 expression facilitated their immortalization by engineering to express the catalytic subunit of telomerase (TERT).  In organotypic culture they stratified and exhibited squamous metaplasia, expressing involucrin and K10.  Thus, hESderK cells proved to be distinct from all three normal p63+ cell types tested. These results indicate that hESderK cells cannot be identified conclusively as keratinocytes or even as ectodermal cells, but may represent an incomplete form of, or deviation from, normal p63+ lineage development. PDF | Back


A keratinocyte hypermotility/growth arrest response involving laminin 5 and p16INK4A activated in wound healing and senescence.

Easwar Natarajan, John D. Omobono II, Zongyou Guo, Susan Hopkinson, Alexander J.F. Lazar, Thomas Brenn, Jonathan C. Jones, and James G. Rheinwald

Department of Dermatology and Department of Pathology, Brigham and Women’s Hospital, Harvard Skin Disease Research Center, Harvard Medical School, Boston, MA, and Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, IL USA

Keratinocytes become migratory to heal wounds, during early neoplastic invasion, and when undergoing telomere-unrelated senescence in culture. All three settings are associated with expression of the cell cycle inhibitor p16INK4A and of the basement membrane protein laminin 5. We have investigated cause-and-effect relationships among laminin 5, p16, hypermotility, and growth arrest. Plating primary human keratinocytes on the 2 precursor form of laminin 5 (LN5’) immediately induced directional hypermotility at ~125 m/hr, followed by p16 expression and growth arrest. Cells deficient in p16 and either p14ARF or p53 became hypermotile in response to LN5’ but did not growth-arrest. Plating on LN5’ triggered smad nuclear translocation and all LN5’ effects were blocked by a TGF receptor I (TGFβRI) kinase inhibitor. In contrast, plating cells on collagen I triggered a TGFβRI kinase-independent hypermotility unaccompanied by smad translocation or growth arrest. Plating on control surfaces with TGF induced hypermotility after a 1d lag time and growth-arrest by a p16-independent mechanism. Keratinocytes serially cultured with TGFβRI kinase inhibitor grew for an extended lifespan and their immortalization by TERT expression was facilitated. These results reveal fundamental features of a keratinocyte hypermotility/growth arrest response that is activated in wound healing, tumor suppression, and during serial culture.
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Immortalized keratinocyte lines derived from human embryonic stem cells.

Iuchi S, Dabelsteen S, Easley K, Rheinwald JG, Green H.

Department of Cell Biology, Harvard Medical School, Boston, MA 02115.

Cells of the human embryonic stem (hES) cell line H9, when cultured in the form of embryoid bodies, give rise to cells with markers of the keratinocyte of stratified squamous epithelia. Keratinocytes also form in nodules produced in scid mice by injected H9 cells; the hES-derived keratinocytes could be recovered in culture, where their colonies underwent a peculiar form of fragmentation. Whether formed from embryoid bodies or in nodules, hES-derived keratinocytes differed from postnatal keratinocytes in their much lower proliferative potential in culture; isolated single keratinocytes could not be expanded into mass cultures. Although their growth was not improved by transduction with the hTERT gene, these keratinocytes were immortalized by transduction with the E6E7 genes of HPV16. Clonally derived lines isolated from E6E7-transduced keratinocytes continued to express markers of the keratinocyte lineage, but the frequency with which they terminally differentiated was reduced compared with keratinocytes cultured from postnatal human epidermis. If other hES-derived somatic cell types also prove to be restricted in growth potential, not identical to the corresponding postnatal cell types, and to require immortalization for clonal isolation and expansion, these properties will have to be considered in planning their therapeutic use. PDF | Back


Co-expression of p16INK4A and laminin 5 by keratinocytes: a wound-healing response coupling hypermotility with growth arrest that goes awry during epithelial neoplastic progression.

Natarajan E, Omobono JD 2nd, Jones JC, Rheinwald JG.

Department of Dermatology and Harvard Skin Disease Research Center, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.

The replicative lifespan of human keratinocytes in culture is restricted by a telomere-unrelated induction of p16INK4A (p16) and p14ARF. We have found that, in vivo, p16 is expressed by epidermal and oral keratinocytes at the migrating fronts of healing wounds and at the stromal interface of severely dysplastic and early invasive lesions and that such cells also invariably display increased expression of Laminin 5 (Lam5). In culture, p16 and Lam5 are coexpressed in keratinocytes at senescence, at the edges of wounds made in confluent cultures, and when cells are plated on dishes coated with the gamma2 precursor form of Lam5 (Lam5gamma2pre). Lam5/p16 coexpression in all three in vitro settings is associated with directional hypermotility and growth arrest. Hypermotility and growth arrest are uncoupled in p16- and p14ARF/p53-deficient keratinocytes and squamous cell carcinoma (SCC) cells; such cells become hypermotile is response to Lam5gamma2pre but do not growth arrest. Thus, the Lam5/p16 response is activated in normal wound healing, causing growth arrest of migratory keratinocytes that lead wound reepithelialization. This response also becomes activated at a critical stage of neoplastic progression, acting as a tumor suppressor mechanism. Rare premalignant cells that lose p16 remain motile and proliferative, thereby resulting in invasive growth as SCC. PDF | Back


Identification of novel candidate oncogenes and tumor suppressors in malignant pleural mesothelioma using large-scale transcriptional profiling.

Gordon GJ, Rockwell GN, Jensen RV, Rheinwald JG, Glickman JN, Aronson JP, Pottorf BJ, Nitz MD, Richards WG, Sugarbaker DJ, Bueno R.

Thoracic Surgery Oncology Laboratory, Division of Thoracic Surgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA. ggordon@partners.org

Malignant pleural mesothelioma (MPM) is a highly lethal, poorly understood neoplasm that is typically associated with asbestos exposure. We performed transcriptional profiling using high-density oligonucleotide microarrays containing approximately 22,000 genes to elucidate potential molecular and pathobiological pathways in MPM using discarded human MPM tumor specimens (n = 40), normal lung specimens (n = 4), normal pleura specimens (n = 5), and MPM and SV40-immortalized mesothelial cell lines (n = 5). In global expression analysis using unsupervised clustering techniques, we found two potential subclasses of mesothelioma that correlated loosely with tumor histology. We also identified sets of genes with expression levels that distinguish between multiple tumor subclasses, normal and tumor tissues, and tumors with different morphologies. Microarray gene expression data were confirmed using quantitative reverse transcriptase-polymerase chain reaction and protein analysis for three novel candidate oncogenes (NME2, CRI1, and PDGFC) and one candidate tumor suppressor (GSN). Finally, we used bioinformatics tools (ie, software) to create and explore complex physiological pathways. Combined, all of these data may advance our understanding of mesothelioma tumorigenesis, pathobiology, or both. PDF | Back


Biochemical and functional analysis of smallpox growth factor (SPGF) and anti-SPGF monoclonal antibodies.

Kim M, Yang H, Kim SK, Reche PA, Tirabassi RS, Hussey RE, Chishti Y, Rheinwald JG, Morehead TJ, Zech T, Damon IK, Welsh RM, Reinherz EL.

Department of Medical Oncology, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA.

Variola, the causative agent of smallpox, is a highly infectious double-stranded DNA virus of the orthopox genus that replicates within the cytoplasm of infected cells. For unknown reasons prominent skin manifestations, including "pox," mark the course of this systemic human disease. Here we characterized smallpox growth factor (SPGF), a protein containing an epidermal growth factor (EGF)-like domain that is conserved among orthopox viral genomes, and investigated its possible mechanistic link. We show that after recombinant expression, refolding, and purification, the EGF domain of SPGF binds exclusively to the broadly expressed cellular receptor, erb-B1 (EGF receptor), with subnanomolar affinity, stimulating the growth of primary human keratinocytes and fibroblasts. High affinity monoclonal antibodies specific for SPGF reveal in vivo immunoprotection in a murine vaccinia pneumonia model by a mechanism distinct from viral neutralization. These findings suggest that blockade of pathogenic factor actions, in general, may be advantageous to the infected host. PDF | Back


Co-expression of p16(INK4A) and laminin 5 gamma2 by microinvasive and superficial squamous cell carcinomas in vivo and by migrating wound and senescent keratinocytes in culture.

Natarajan E, Saeb M, Crum CP, Woo SB, McKee PH, Rheinwald JG.

Department of Dermatology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.

Am J Pathol. 2003 Aug;163(2):477-91.

The high frequency of mutation, deletion, and promoter silencing of the gene encoding p16(INK4A) (p16) in premalignant dysplasias and squamous cell carcinomas (SCC) of epidermis and oral epithelium classifies p16 as a tumor suppressor. However, the point during neoplastic progression at which this protein is expressed and presumably impedes formation of an SCC is unknown. Induction of p16 has been found to be responsible for the senescence arrest of normal human keratinocytes in culture, suggesting the possibility that excessive or spatially abnormal cell growth in vivo triggers p16 expression. We examined 73 skin and oral mucosal biopsy specimens immunohistochemically to test this hypothesis. p16 was not detectable in benign hyperplastic lesions, but instead was expressed heterogeneously in some dysplastic and carcinoma in situ lesions and consistently at areas of microinvasion and at superficial margins of advanced SCCs. p16-positive cells in these regions coexpressed the gamma2 chain of laminin 5, identified previously as a marker of invasion in some carcinomas. Normal keratinocytes undergoing senescence arrest in culture proved to coordinately express p16 and gamma2 and this was frequently associated with increased directional motility. Keratinocytes at the edges of wounds made in confluent early passage cultures also coexpressed p16 and gamma2, accompanying migration to fill the wound. These results have identified the point during neoplastic progression in stratified squamous epithelial at which the tumor suppressor p16 is expressed and suggest that normal epithelia may use the same mechanism to generate non-dividing, motile cells for wound repair. PDF | Back


Selective suppression of monocyte chemoattractant protein-1 expression by human papillomavirus E6 and E7 oncoproteins in human cervical epithelial and epidermal cells.

Kleine-Lowinski K, Rheinwald JG, Fichorova RN, Anderson DJ, Basile J, Munger K, Daly CM, Rosl F, Rollins BJ.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA.

Int J Cancer. 2003 Nov 10;107(3):407-15.

Infection of cervical keratinocytes by high-risk HPV is involved in the etiology of cervical carcinoma. Since viral products are immunogenic, development of cancer may require suppression of immune responses directed against infected epithelial cells. Many markers of host immune effector responses decrease as cervical intraepithelial neoplasia progresses. Among these is epithelial cell expression of the chemokine MCP-1, though the mechanism for its suppression is unclear. Here, we show that the E6 and E7 viral oncogenes from high-risk HPV, individually and together, suppress MCP-1 expression in primary epithelial cells derived from the female genital tract. This is not a consequence of global suppression of chemokine expression since other chemokines, including IP-10, IL-8 and RANTES, were less affected. Furthermore, 4 of 6 HPV-positive cervical carcinoma cell lines did not express MCP-1. Our data indicate that suppression of MCP-1 expression is part of the program of high-risk HPV E6/E7-induced transformation of primary epithelial cells. These observations are consistent with a model in which MCP-1 expression by infected keratinocytes, which would stimulate an immune attack on HPV-transformed cells, is suppressed for invasive cervical cancer to appear. PDF | Back


A two-stage, p16(INK4A)- and p53-dependent keratinocyte senescence mechanism that limits replicative potential independent of telomere status.

Rheinwald JG, Hahn WC, Ramsey MR, Wu JY, Guo Z, Tsao H, De Luca M, Catricala C, O'Toole KM.

Department of Dermatology, Brigham and Women's Hospital, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA. JRheinwald@rics.bwh.harvard.edu

Mol Cell Biol. 2002 Jul;22(14):5157-72.

With increasing frequency during serial passage in culture, primary human keratinocytes express p16(INK4A) (p16) and undergo senescence arrest. Keratinocytes engineered to express hTERT maintain long telomeres but typically are not immortalized unless, by mutation or other heritable event, they avoid or greatly reduce p16 expression. We have confirmed that keratinocytes undergo p16-related senescence during growth in culture, whether in the fibroblast feeder cell system or in the specialized K-sfm medium formulation, and that this mechanism can act as a barrier to immortalization following hTERT expression. We have characterized the p16-related arrest mechanism more precisely by interfering specifically with several regulators of cell cycle control. Epidermal, oral mucosal, corneal limbal, and conjunctival keratinocytes were transduced to express a p16-insensitive mutant cdk4 (cdk4(R24C)), to abolish p16 control, and/or a dominant negative mutant p53 (p53DD), to abolish p53 function. Expression of either cdk4(R24C) or p53DD alone had little effect on life span, but expression of both permitted cells to divide 25 to 43 population doublings (PD) beyond their normal limit. Keratinocytes from a p16(+/-) individual transduced to express p53DD alone displayed a 31-PD life span extension associated with selective growth of variants that had lost the wild-type p16 allele. Cells in which both p53 and p16 were nonfunctional divided rapidly during their extended life span but experienced telomere erosion and ultimately ceased growth with very short telomeres. Expression of hTERT in these cells immortalized them. Keratinocytes engineered to express cdk4(R24C) and hTERT but not p53DD did not exhibit an extended life span. Rare immortal variants exhibiting p53 pathway defects arose from them, however, indicating that the p53-dependent component of keratinocyte senescence is telomere independent. Mutational loss of p16 and p53 has been found to be a frequent early event in the development of squamous cell carcinoma. Our results suggest that such mutations endow keratinocytes with extended replicative potential which may serve to increase the probability of neoplastic progression. PDF | Back


Novel p53 splice site mutations in three families with Li-Fraumeni syndrome.

Verselis SJ, Rheinwald JG, Fraumeni JF Jr, Li FP.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, MA 02115, USA.

Oncogene. 2000 Aug 31;19(37):4230-5.

Germline mutations in the p53 tumor suppressor gene predispose to a variety of cancers in families with Li-Fraumeni syndrome. Most germline p53 mutations observed to date cause amino acid substitutions in the protein's central sequence-specific DNA binding domain. Outside this conserved core region, however, we found novel alterations in sequences that regulate precursor mRNA splicing in three Li-Fraumeni syndrome families. Two splice site mutations affected the consensus sequence at the splice donor sites of introns 1 and 9, and produced unstable variant transcripts in normal cells. A third mutation at the splice acceptor site of intron 9 generated splicing at a cryptic acceptor site in intron 9. These splice site alterations emphasize the need to examine both noncoding and untranslated regions of the p53 gene for germline mutations in Li-Fraumeni syndrome families. PDF | Back


Human keratinocytes that express hTERT and also bypass a p16(INK4a)-enforced mechanism that limits life span become immortal yet retain normal growth and differentiation characteristics.

Dickson MA, Hahn WC, Ino Y, Ronfard V, Wu JY, Weinberg RA, Louis DN, Li FP, Rheinwald JG.

Division of Dermatology, Department of Medicine and Harvard Skin Disease Research Center, Brigham and Women's Hospital, Boston, Massachusetts, USA.

Mol Cell Biol. 2000 Feb;20(4):1436-47.

Normal human cells exhibit a limited replicative life span in culture, eventually arresting growth by a process termed senescence. Progressive telomere shortening appears to trigger senescence in normal human fibroblasts and retinal pigment epithelial cells, as ectopic expression of the telomerase catalytic subunit, hTERT, immortalizes these cell types directly. Telomerase expression alone is insufficient to enable certain other cell types to evade senescence, however. Such cells, including keratinocytes and mammary epithelial cells, appear to require loss of the pRB/p16(INK4a) cell cycle control mechanism in addition to hTERT expression to achieve immortality. To investigate the relationships among telomerase activity, cell cycle control, senescence, and differentiation, we expressed hTERT in two epithelial cell types, keratinocytes and mesothelial cells, and determined the effect on proliferation potential and on the function of cell-type-specific growth control and differentiation systems. Ectopic hTERT expression immortalized normal mesothelial cells and a premalignant, p16(INK4a)-negative keratinocyte line. In contrast, when four keratinocyte strains cultured from normal tissue were transduced to express hTERT, they were incompletely rescued from senescence. After reaching the population doubling limit of their parent cell strains, hTERT(+) keratinocytes entered a slow growth phase of indefinite length, from which rare, rapidly dividing immortal cells emerged. These immortal cell lines frequently had sustained deletions of the CDK2NA/INK4A locus or otherwise were deficient in p16(INK4a) expression. They nevertheless typically retained other keratinocyte growth controls and differentiated normally in culture and in xenografts. Thus, keratinocyte replicative potential is limited by a p16(INK4a)-dependent mechanism, the activation of which can occur independent of telomere length. Abrogation of this mechanism together with telomerase expression immortalizes keratinocytes without affecting other major growth control or differentiation systems. PDF | Back


Human sweat gland myoepithelial cells express a unique set of cytokeratins and reveal the potential for alternative epithelial and mesenchymal differentiation states in culture.

Schon M, Benwood J, O'Connell-Willstaedt T, Rheinwald JG.

Division of Dermatology/Department of Medicine, Brigham and Women's Hospital, and Division of Cell Growth and Regulation, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA.

J Cell Sci. 1999 Jun;112 ( Pt 12):1925-36.

We have characterized precisely the cytokeratin expression pattern of sweat gland myoepithelial cells and have identified conditions for propagating this cell type and modulating its differentiation in culture. Rare, unstratified epithelioid colonies were identified in cultures initiated from several specimens of full-thickness human skin. These cells divided rapidly in medium containing serum, epidermal growth factor (EGF), and hydrocortisone, and maintained a closely packed, epithelioid morphology when co-cultured with 3T3 feeder cells. Immunocytochemical and immunoblot analysis disclosed that the cells differed from keratinocytes in that they were E-cadherin-negative, vimentin-positive, and expressed an unusual set of cytokeratins, K5, K7, K14, and K17. When subcultured without feeder cells, they converted reversibly to a spindle morphology and ceased K5 and K14 expression. Under these conditions, EGF deprivation induced flattening, growth arrest, and expression of alpha-smooth muscle actin ((&agr;)-sma). Coexpression of keratins and alpha-sma is a hallmark of myoepithelial cells, a constituent of secretory glands. Immunostaining of skin sections revealed that only sweat gland myoepithelial cells expressed the same pattern of keratins and alpha-sma and lack of E-cadherin as the cell type we had cultured. Interestingly, our immunocytochemical analysis of ndk, a skin-derived cell line of uncertain identity, suggests that this line is of myoepithelial origin. Earlier immunohistochemical studies by others had found myoepithelial cells to be K7-negative. We tested five K7-specific antibodies that can recognize this protein in western blots and in the assembled keratin filaments of mesothelial cells. Three of these antibodies did not recognize the K7 present in myoepithelial cell filaments or in HeLa cell filaments, indicating that some K7 epitopes are masked when K7 pairs with K17 instead of with its usual keratin filament partner, K19. PDF | Back


Generation of papillomavirus-immortalized cell lines from normal human ectocervical, endocervical, and vaginal epithelium that maintain expression of tissue-specific differentiation proteins.

Fichorova RN, Rheinwald JG, Anderson DJ.

Fearing Research Laboratory, Boston, MA 02115, USA.

Biol Reprod. 1997 Oct;57(4):847-55.

We have established and characterized three cell lines from normal human vaginal, ectocervical, and endocervical epithelia immortalized by expression of human papillomavirus 16/E6E7. The lines (VK2/E6E7, Ect1/E6E7, and End1/E6E7) displayed distinctive morphologies at the level of light microscopy when cultured in calcium-supplemented (0.4 mM) keratinocyte serum-free medium and maintained a stable phenotype after more than 1 yr of continuous passage. They were compared to primary cell cultures and epithelial cells in sections of the respective native tissues for expression of epithelial differentiation proteins. All cell lines expressed cytokeratin (CK) 8, CK18, and CK19, and some cells in all three cell lines expressed CK16, involucrin, and the secretory component of the polymeric immunoglobulin receptor. The vaginal and ectocervical cell lines expressed CK10 and CK13, whereas the endocervical line did not. With the exception of CK8 and CK18 expression, the morphological and immunocytochemical characteristics of the immortalized lines closely resembled those of their respective tissues of origin and primary cultures, and all differed significantly from the HeLa cervical adenocarcinoma cell line. These new cell lines may provide the basis for valid, reproducible in vitro models for studies on cervicovaginal physiology and infections and for testing pharmacological agents for intravaginal application. PDF | Back


Intraperitoneal injection of genetically modified, human mesothelial cells for systemic gene therapy.

Murphy JE, Rheinwald JG.

Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.

Hum Gene Ther. 1997 Nov 1;8(16):1867-79.

An ideal cell type for ex vivo gene therapy should be easy to biopsy, propagate, and genetically engineer in culture, should be transplantable using simple procedures, and should express therapeutic proteins at useful levels. The mesothelial cell appears to satisfy these criteria. Several thousand proliferative mesothelial cells were present in typical specimens of nonpathologic human peritoneal fluid obtained by needle aspiration. These divided rapidly in a specialized medium to yield pure cultures of approximately 10(7) cells within 2 weeks. The replicative lifespan of mesothelial cells cultured from adults was approximately 42-52 population doublings, permitting expansion and cryopreservation of a lifetime supply of autologous cells from one fluid sample. Cells transduced with a human growth hormone (hGH) adenoviral vector secreted 100-300 microg of hGH/10(6) cells per day for at least 6 weeks in culture when maintained at quiescence. Intraperitoneal injection of transduced cells into athymic mice resulted in rapid systemic delivery of hGH, with peak plasma levels of 0.1-1 microg/ml declining over 3 weeks to <1 ng/ml. Mice receiving a second injection of engineered cells displayed the same plasma hGH levels and duration as naive mice. Cells labeled with a beta-galactosidase vector were identifiable by in situ enzymatic staining as clusters attached to peritoneal surfaces at multiple sites for at least 19 days after injection. Cells serially passaged through about three-quarters of their lifespan before transduction and injection were as effective at hGH delivery as earlier-passage cells. These results indicate the clinical potential for ex vivo gene therapy using mesothelial cells. PDF | Back


Hyaluronic acid capsule and the role of streptococcal entry into keratinocytes in invasive skin infection.

Schrager HM, Rheinwald JG, Wessels MR.

Channing Laboratory, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.

J Clin Invest. 1996 Nov 1;98(9):1954-8.

It has been suggested that entry of pathogenic bacteria, including streptococci, into epithelial cells may represent an early stage of invasive infections. We found that poorly encapsulated wild-type strains and unencapsulated mutants of group A Streptococcus entered cultured human keratinocytes with high efficiency, while strains that produced large amounts of hyaluronic acid capsule did not, regardless of M-protein type or clinical source of the isolate. However, encapsulated streptococci produced extensive local necrosis and systemic infection in a mouse model of skin infection, while an isogenic acapsular strain did not. The results implicate the hyaluronic acid capsule as a virulence factor in soft tissue infection. Entry of poorly encapsulated group A Streptococcus into human epithelial cells does not appear to represent an initial step in invasive disease; rather, the capacity of encapsulated strains to avoid uptake by epithelial cells is associated with enhanced virulence in skin and soft tissue infection. PDF | Back


A limited role for retinoic acid and retinoic acid receptors RAR alpha and RAR beta in regulating keratin 19 expression and keratinization in oral and epidermal keratinocytes.

Schon M, Rheinwald JG.

Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.

J Invest Dermatol. 1996 Sep;107(3):428-38.

Different types of stratified squamous epithelia-for example, the "orthokeratinized" epidermis, the "parakeratinized" gingiva, and the "nonkeratinized" oral lining mucosal epithelia-are formed by intrinsically distinct keratinocyte subtypes. These subtypes exhibit characteristic patterns of keratin protein expression in vivo and in culture. Keratin 19 is an informative subtype-specific marker because the basal cells of only nonkeratinizing epithelia express K19 in vivo and in culture. Epidermal keratinocytes normally do not express K19, but can be induced to do so in culture by retinoic acid (RA). Keratinocyte subtypes express the retinoic acid receptor (RAR) beta at levels roughly correlated with their level of K19 expression in culture and their potential for forming a nonkeratinized epithelium in vivo. We tested the hypothesis that the level of RAR beta expressed by a keratinocyte determines its K19 expression and its form of suprabasal differentiation. Normal human epidermal and gingival keratinocytes stably overexpressing either RAR beta or RAR alpha were generated by defective retroviral transduction. Overexpression of either receptor enhanced the RA inducibility of K19 in conventional culture, in that the proportion of the transductants becoming K19+ in response to RA was markedly increased compared with controls. The pattern of differentiation of the epithelium formed in organotypic culture, assessed by basal K19 and suprabasal K1, K4, and filaggrin expression, however, was unaltered by RAR overexpression. Thus, the susceptibility of keratinocytes to regulation of K19 expression by retinoids is conditional, and levels of neither RAR beta nor RAR alpha are limiting to the intrinsic mechanism that specifies alternate differentiation pathways for stratified squamous epithelia. PDF | Back


The Wilms tumour gene WT1 is expressed in murine mesoderm-derived tissues and mutated in a human mesothelioma.

Park S, Schalling M, Bernard A, Maheswaran S, Shipley GC, Roberts D, Fletcher J, Shipman R, Rheinwald J, Demetri G, et al.

Massachusetts General Hospital Cancer Center, Charlestown 02129.

Nat Genet. 1993 Aug;4(4):415-20.

The tumour suppressor gene WT1 encodes a transcription factor expressed in tissues of the genito-urinary system. Inactivation of this gene is associated with the development of Wilms tumour a pediatric kidney cancer. We show that WT1 is also expressed at high levels in many supportive structures of mesodermal origin in the mouse. We also describe a case of adult human mesothelioma, a tumour derived from the peritoneal lining, that contains a homozygous point mutation within WT1. This mutation, within the putative transactivation domain, converts the protein from a transcriptional repressor of its target sequence to a transcriptional activator. The role of WT1 in normal development thus extends to diverse structures derived from embryonic mesoderm and disruption of WT1 function contributes to the onset of adult, as well as pediatric, tumours. PDF | Back


In vitro propagation of human ocular surface epithelial cells for transplantation.

Lindberg K, Brown ME, Chaves HV, Kenyon KR, Rheinwald JG.

Department of Research and Development, BioSurface Technology, Inc., Cambridge, Massachusetts 02139.

Invest Ophthalmol Vis Sci. 1993 Aug;34(9):2672-9.

PURPOSE. To examine the possibility that ocular surface epithelial cells might be grown in culture for use as grafts. METHODS. The proliferative capacity of epithelial cells cultured from the conjunctiva, limbus, and central cornea of normal human eyes was compared. Single cells disaggregated from approximately 1 mm2 biopsy specimens were serially cocultured with lethally irradiated mouse 3T3 fibroblasts. To study the cells' ability to reform a stratified epithelium, confluent limbal cultures were released as an intact cell sheet with the enzyme Dispase and transplanted to a dermal connective tissue bed in nude mice. Attachment and differentiation properties of the reconstituted epithelium were examined immunohistochemically. RESULTS. Central corneal epithelial cells could not be propagated; they senesced in first or second passage. In contrast, limbal epithelial cells exhibited a substantial (i.e., mean of 23 population doublings) and conjunctival cells a moderate (i.e., mean of 11 population doublings) proliferative capacity. Within 4 days of transplantation to the nude mouse dermis, cultured limbal epithelial cells formed an epithelium 5-6 cell layers thick. The epithelium adhered firmly to the graft bed, and deposition of the basement membrane and anchoring fibril protein collagens IV and VII and laminin was detectable immunohistochemically. The transplanted epithelium displayed limbuslike compartmental expression of keratins K3, K13, and K19, and of the enzyme enolase. CONCLUSIONS. These results support the concept that corneal epithelial stem cells are located in the limbus and indicate that cultured autologous limbal cells may function as grafts to permanently restore the corneal epithelium after severe ocular surface injury. PDF | Back


Abnormal expression of retinoic acid receptors and keratin 19 by human oral and epidermal squamous cell carcinoma cell lines.

Hu L, Crowe DL, Rheinwald JG, Chambon P, Gudas LJ.

Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts.

Cancer Res. 1991 Aug 1;51(15):3972-81.

We have analyzed the expression of the three retinoic acid receptor (RAR) (alpha, beta, gamma) mRNAs and the intermediate filament protein keratin 19 (K19) mRNA in cell lines cultured from oral and epidermal human squamous cell carcinoma (SCC) and from benign, hyperplastic, and hyperkeratotic (leukoplakia) lesions arising in various regions of the oral cavity. Seven of the SCC lines were derived from tumors arising in regions of the oral cavity in which the normal epithelial cells (keratinocytes) express RAR beta transcripts. Seven of the nine SCC lines tested did not exhibit detectable RAR beta mRNA levels, even in response to addition of retinoic acid (RA). The RAR beta gene did not appear to be rearranged or deleted in the five nonexpressing SCC lines examined by Southern analysis. The steady-state RAR gamma mRNA levels were 2- to 4-fold lower in 6 of the 9 SCC lines than in their normal counterparts, whereas the RAR alpha message levels in SCC lines were similar to those of the normal cell strains. The expression of keratin 19 message, which is RA inducible in normal keratinocytes, was also abnormal in many of the SCC cell lines. Some SCC lines, e.g., those derived form tumors of the soft palate epithelium, did not express high levels of K19 message even though normal soft palate keratinocytes expressed high levels of K19 mRNA. Two of the nine SCC lines expressed higher than normal levels of K19 mRNA, and this expression was RA independent. Cells cultured from four oral leukoplakia lesions were also examined and found to express RAR beta mRNA at relatively normal levels, but they expressed RAR gamma message at half the level of epithelial cells cultured from normal tissue. These results show that the correlation between RAR beta gene expression and K19 gene expression that we have observed in the various normal keratinocyte subtypes of the oral cavity (D.L. Crowe et al., manuscript in preparation) is not present in transformed keratinocytes (SCC cells). The lack of apparent RA regulation of the K19 gene in SCC lines may be associated with other aberrations in differentiation which have been identified in SCC cells. Abnormally low expression of the RAR beta receptor may contribute to neoplastic progression in stratified squamous epithelia. It may also determine whether a tumor is responsive to RA as a chemotherapeutic agent. PDF | Back


Variable expression of retinoic acid receptor (RAR beta) mRNA in human oral and epidermal keratinocytes; relation to keratin 19 expression and keratinization potential.

Crowe DL, Hu L, Gudas LJ, Rheinwald JG.

Division of Cell Growth and Regulation, Dana-Farber Cancer Institute, Boston, MA 02115.

Differentiation. 1991 Dec;48(3):199-208.

Previous studies have revealed that the cells that form the different regions of the oral and epidermal stratified squamous epithelia represent a number of intrinsically distinct keratinocyte subtypes, each of which is developmentally programmed to preferentially express a particular pattern of keratins and type of suprabasal histology. Retinoic acid (RA) is known to modulate stratified squamous epithelial differentiation, including expression of the basal cell keratin K19 and the suprabasal keratins K1/K10 and K4/K13. We have found that all keratinocyte subtypes are similar in their steady state levels of RAR alpha and RAR gamma mRNAs in culture and that these levels are only minimally affected by RA. In contrast, RAR beta mRNA expression varies greatly among keratinocyte subtypes and, in eight of ten cell strains examined, directly correlated with their levels of K19 mRNA. Exposure to 10(-6) M RA increases the levels of RAR beta and K19 mRNA; conversely, complete removal of RA from the medium results in reduced levels of these messages. RA does not coordinately induce RAR beta and K19 messages in nonkeratinocyte cell types: fibroblasts cultured in the presence of 10(-6) M RA express very high levels of RAR beta mRNA but do not express detectable K19, and mesothelial cells decrease their levels of RAR beta and K19 mRNA in response to 10(-6) M RA. The correlation between RAR beta and K19 mRNA levels in most keratinocyte subtypes suggests a role for RAR beta in specifying patterns of keratin expression and suprabasal differentiation in stratified squamous epithelia. PDF | Back


Three distinct keratinocyte subtypes identified in human oral epithelium by their patterns of keratin expression in culture and in xenografts.

Lindberg K, Rheinwald JG.

Division of Cell Growth and Regulation, Dana-Farber Cancer Institute, Boston, MA.

Differentiation. 1990 Dec;45(3):230-41.

We have characterized the cells that form the human oral epithelia by analyzing their patterns of keratin expression in culture and in transplants. Keratinocytes of all oral regions synthesized high levels of keratins K5/K14 and K6/K16,K17, as expressed by cells of all stratified squamous epithelia in culture. However, cells from different regions varied in their expression in culture of retinoid-inducible (K19 and K13) and simple epithelial (K7, K8 and K18) keratins. By these criteria, all oral cells could be classified as belonging to one of three intrinsically distinct subtypes: "keratinizing" (gingiva, hard palate), "typical nonkeratinizing" (inner cheek, floor of mouth, ventral tongue) and "special non-keratinizing" (soft palate), all of which differed from the epidermal keratinocyte subtype. Cells from fetal floor of mouth expressed a pattern of keratins in culture markedly different from that of adult floor of mouth cells but identical to that of the adult "special nonkeratinizing" subtype and similar to that of several oral squamous cell carcinoma lines. When cultures of oral keratinocytes were grafted to the dermis of nude mice, they formed stratified epithelial structures after 10 days. In some areas of the stratified structures, the basal layer recapitulated the K19 expression pattern of the oral region from which they had originated. Thus, regional differentiation of the oral epithelium is based on an intrinsic specialization of regional keratinocyte stem cells. Additionally, oral cell transformation either frequently involves reversion to the fetal keratin program or else oral cells that express this keratin program are especially susceptible to transformation. PDF | Back


Specific effects of ras oncogene expression on the growth and histogenesis of human epidermal keratinocytes.

Henrard DR, Thornley AT, Brown ML, Rheinwald JG.

Division of Cell Growth and Regulation, Dana-Farber Cancer Institute, Boston, Massachusetts.

Oncogene. 1990 Apr;5(4):475-81.

Little progress has been made in identifying specific regulatory pathways that might be affected in cells by a mutationally activated p21ras when its expression does not lead to complete transformation. We wished to determine whether a normal, diploid human epithelial cell in which activation of ras had occurred could be identified in culture and, furthermore, whether expression of a mutant p21ras in such an otherwise normal cell would result in abnormal histogenic behavior in vivo. Thus, we introduced the v-Ha-ras gene into an early passage culture of normal human epidermal keratinocytes via a defective retrovirus. We examined these genetically engineered cells for changes in growth and differentiation, both in culture and in the epithelium formed when cultures were grafted to the skin of nude mice. We have found that keratinocytes expressing p21v-ras are independent of epidermal growth factor (EGF)--a factor which is normally essential for progressive colony growth, but that they are otherwise indistinguishable in culture from normal cells. v-ras keratinocytes also secrete a factor possessing some specific biological activities of members of the fibroblast growth factor (FGF) family, but which is distinct from acidic and basic FGF. In short-term dermal grafts the v-ras cells form a non-invasive and normally differentiating epidermis. However, the cells express elevated levels of keratin 19, which is a characteristic of fetal epidermis and of premalignant lesions of some stratified squamous epithelia. PDF | Back


Expression of ras oncogenes in cultured human cells alters the transcriptional and posttranscriptional regulation of cytokine genes.

Demetri GD, Ernst TJ, Pratt ES 2nd, Zenzie BW, Rheinwald JG, Griffin JD.

Division of Tumor Immunology, Dana-Farber Cancer Institute, Boston, Massachusetts.

J Clin Invest. 1990 Oct;86(4):1261-9.

Autonomous production of cytokines such as the hematopoietic colony-stimulating factors (CSFs), IL-1, or IL-6 has been demonstrated in numerous human and murine neoplasms, and may be involved in the pathogenesis of several paraneoplastic syndromes such as leukocytosis, fever, and hypercalcemia. Because of the high frequency with which mutations in ras protooncogenes have been detected in human tumors, as well as evidence linking ras gene products to activation of certain cellular functions, we investigated whether ras mutations might influence the regulation of cytokine genes. Normal human fibroblasts transfected with a mutant val12 H-ras oncogene expressed increased levels of mRNA transcripts encoding granulocyte-CSF (G-CSF), granulocyte-macrophage-CSF (GM-CSF), and IL-1 beta compared with controls. Human mesothelioma cells transfected with a mutant asp12 N-ras oncogene exhibited similar alterations in cytokine gene expression. Estimates of transcriptional activity by nuclear run-on analysis revealed a selective increase in transcription only for the IL-1 gene. Analysis of mRNA half-life demonstrated a marked increase in the stability of numerous cytokine transcripts, including G-CSF, GM-CSF, IL-1, and IL-6. The addition of anti-IL-1 neutralizing antibody to cultures of cells expressing ras mutants did not block the expression of any of the cytokines examined, suggesting that the baseline expression of GM-CSF, G-CSF, and IL-6 was not a secondary event due to the increased transcription of IL-1. These results indicate that mutations in ras genes may alter expression of several cytokine genes through both transcriptional and posttranscriptional mechanisms. PDF | Back


Environment-dependent growth inhibition of human epidermal keratinocytes by recombinant human transforming growth factor-beta.

Rollins BJ, O'Connell TM, Bennett G, Burton LE, Stiles CD, Rheinwald JG.

Division of Medicine, Dana-Farber Cancer Institute, Boston, MA.

J Cell Physiol. 1989 Jun;139(3):455-62.

Transforming growth factor-beta (TGF-beta) purified from platelets is a potent growth inhibitor of several normal epithelial cell types in culture. In contrast, some carcinoma cell lines derived from tumors of these same tissues are resistant to this factor. Using recombinant human TGF-beta, the authors have confirmed these results with six normal human epidermal keratinocyte strains and four human epidermal squamous carcinoma cell lines. However, the sensitivity of normal cells to TGF-beta was found to depend on the culture conditions. When grown in a specialized nutrient medium supplemented with pituitary extract, keratinocytes were completely inhibited by the addition of 0.3 ng/ml TGF-beta. In contrast, when their growth was supported by cocultivation with 3T3 fibroblast feeder cells, 30- to 100-fold higher concentrations of TGF-beta were required to achieve comparable growth inhibition. This differential sensitivity occurred despite the fact that in both culture systems TGF-beta in the culture medium had a half-life of about 50 minutes, becoming tightly bound to the surface of the culture dish. Bound TGF-beta proved to be biologically active and stable for about a week in the absence of 3T3 feeder cells. Incubating 3T3 cells on TGF-beta-coated dishes, however, resulted in nearly quantitative removal and degradation of the TGF-beta within 2 days, permitting normal rates of keratinocyte growth. The binding of TGF-beta to surfaces and the ability of fibroblasts to attenuate its inhibitory activity for epithelial cells must be considered when evaluating in vitro models and in planning strategies for the use of this factor in vivo. PDF | Back


Suprabasal 40 kd keratin (K19) expression as an immunohistologic marker of premalignancy in oral epithelium.

Lindberg K, Rheinwald JG.

Department of Oral Pathology and Oral Medicine, Harvard School of Dental Medicine, Boston, Massachusetts.

Am J Pathol. 1989 Jan;134(1):89-98.

The authors have studied the expression of keratin 19 in normal oral mucosa and in oral lesions exhibiting a range of histopathologic changes that are thought to precede squamous cell carcinoma. Formalin-fixed, paraffin-embedded sections were pretreated with pronase and stained with a K19-specific antibody by the avidin-biotin immunoperoxidase method. In nonkeratinized mucosa, whether normal or benign hyperplastic, K19 was detectable in the basal cell layer. In keratinized mucosa, whether normal or benign hyperplastic, there was no detectable K19. All lesions from any oral site that exhibited atypia diagnosed from hematoxylin and eosin stained sections as moderate-to-severe dysplasia or carcinoma in situ, whether hyperkeratotic or not, stained strongly for K19 in the basal and suprabasal cell layers. The number of cell layers that were K19-positive correlated with the level in the epithelium to which dysplasia persisted. Suprabasal K19 staining tended to occur in regions of the epithelium in which expression of the terminal differentiation protein involucrin was delayed or absent. Thus, K19 expression may be linked to the retention of stem cell character or a state otherwise uncommitted to terminal squamous differentiation. Suprabasal K19 staining is clearly correlated with premalignant change in oral epithelium and therefore promises to be a useful tool in oral histopathologic diagnosis. PDF | Back


Expression of colony-stimulating factor genes by normal human mesothelial cells and human malignant mesothelioma cells lines in vitro.

Demetri GD, Zenzie BW, Rheinwald JG, Griffin JD.

Division of Tumor Immunology, Dana-Farber Cancer Institute, Boston, MA 02115.

Blood. 1989 Aug 15;74(3):940-6.

We investigated normal human mesothelial cells and human malignant mesothelioma cell lines for the ability to produce hematopoietic colony-stimulating factors (CSFs) in culture. Early passage cultures of normal diploid human mesothelial cells spontaneously expressed detectable levels of M-CSF mRNA transcripts, but lacked detectable transcripts for GM-CSF or G-CSF. Exposure of normal mesothelial cells to epidermal growth factor (EGF), lipopolysaccharide (LPS), or tumor necrosis factor (TNF) induced expression of G-CSF mRNA. The combination of EGF and TNF induced threefold more G-CSF transcripts than did either factor alone. GM-CSF transcripts were induced only by the combination of TNF and EGF. Interleukin-1 beta (IL-1 beta) transcripts were induced by EGF, TNF, or LPS and were inhibited by hydrocortisone (HC). All malignant mesothelioma cell lines tested also spontaneously expressed M-CSF transcripts. However, in contrast to normal mesothelial cells, two of four malignant mesothelioma cell lines also autonomously expressed G-CSF and GM-CSF transcripts without TNF, EGF, or LPS stimulation. Secretion of biologically active CSFs was confirmed by testing media conditioned by the various cell types examined. The detection of biologically active CSFs correlated well with the presence of detectable CSF transcripts by Northern analysis. These data indicate that (a) normal human mesothelial cells spontaneously express detectable levels of M-CSF mRNA in culture; (b) EGF is an essential cofactor for optimal induction of G-CSF and GM-CSF expression; (c) exposure of normal mesothelial cells to inflammatory mediators such as LPS and TNF increases the levels of transcripts for CSFs and IL-1 beta; and (d) as compared with normal human mesothelial cells, some cell lines of human malignant mesothelioma exhibit aberrant gene expression for multiple cytokines, including G-CSF, GM-CSF, IL-1 beta, and IL-6. PDF | Back


Cloned cDNA sequence for the human mesothelial protein 'mesosecrin' discloses its identity as a plasminogen activator inhibitor (PAI-1) and a recent evolutionary change in transcript processing.

Cicila GT, O'Connell TM, Hahn WC, Rheinwald JG.

Division of Cell Growth and Regulation, Dana-Farber Cancer Institute, Boston, MA.

J Cell Sci. 1989 Sep;94 ( Pt 1):1-10.

Mesosecrin, a Mr approximately 46 x 10(3) glycoprotein secreted in abundance by human mesothelial cells in culture, was recently described by this laboratory. We isolated partial cDNA clones for mesosecrin from a human mesothelial cell cDNA library in lambda gt11 using a specific antiserum. Comparison of mesosecrin cDNA sequences with the recently published sequence for plasminogen activator inhibitor-1 (PAI-1) cloned from cDNA libraries of endothelial and other cell types revealed that mesosecrin and PAI-1 are the same protein. Reverse fibrin autography of electrophoretically fractionated medium from mesothelial cell cultures confirmed that mesosecrin is functional as a plasminogen activator inhibitor. The mesosecrin/PAI-1 cDNA clones hybridized to abundant 3.6 and 2.6 kb (kb = 10(3) bases) mRNAs on Northern blots of cultured human mesothelial cell and endothelial cell RNA. These mRNA sizes correspond to those recently published for human endothelial and fibrosarcoma PAI-1 mRNA, which most likely result from alternate polyadenylation sites. Messages 3.6 and 2.6 kb long were also detected in cells cultured from orangutans and African green monkeys, but only an approximately 3.6 kb mRNA was detected in cells of lower primates and several other mammalian species. Thus the extra polyadenylation site in the PAI-1 gene, responsible for the shorter form of the RNA, apparently has been acquired recently during primate evolution. Because they are more easily propagated in culture than endothelial cells, human mesothelial cells offer a new and advantageous system for PAI-1 production and study of its regulation and function. PDF | Back


Normal human mesothelial cells and fibroblasts transfected with the EJras oncogene become EGF-independent, but are not malignantly transformed.

Tubo RA, Rheinwald JG.

Division of Cell Growth and Regulation, Dana-Farber Cancer Institute, Boston, MA 02115.

Oncogene Res. 1987 Sep-Oct;1(4):407-21.

The nature of the lesion in growth control exerted by the cancer-derived c-H-ras mutation, EJ-ras, and its transforming potential in diploid cells are both poorly understood. We introduced EJ-ras into normal, diploid human mesothelial cells and fibroblasts and obtained transfectants expressing p21EJ-ras. All clones examined were independent of EGF for rapid growth, and all secreted an EGF-like mitogen into the medium at levels sufficient to satisfy the EGF requirement of normal cells. The EJ-ras transfectants were not altered with respect to any other growth requirement, and they were not transformed. Eleven clones tested all retained a finite replicative lifespan which, in most cases, was the same as that of the parent cell strain. Three transfectants tested were not tumorigenic in nude mice. Thus p21EJ-ras can circumvent an important mitogenic signal pathway in human cells. Nevertheless, neither the secretion of an autocrine growth factor nor any other effect of p21EJ-ras serves to malignantly transform normal human cells, in contrast to the susceptibility of some established rodent cell lines to transformation by these mechanisms. PDF | Back


Mesosecrin: a secreted glycoprotein produced in abundance by human mesothelial, endothelial, and kidney epithelial cells in culture.

Rheinwald JG, Jorgensen JL, Hahn WC, Terpstra AJ, O'Connell TM, Plummer KK.

J Cell Biol. 1987 Feb;104(2):263-75.

Human mesothelial cells, endothelial cells, and type II kidney epithelial cells growing in culture devote approximately 3% of their total protein synthesis to the production of an Mr approximately 46-kD, pI 7.1, secreted glycoprotein (designated Sp46). Fibroblasts make about 1/10th as much Sp46 as these cell types, and their synthesis is dependent upon hydrocortisone. Keratinocytes, urothelial cells, conjunctival epithelial cells, and mammary epithelial cells do not make detectable amounts of Sp46. Mesothelial cells secrete Sp46 onto the substratum, and from there it is subsequently released into the medium. Immunofluorescence analysis using specific antisera discloses that Sp46 is deposited beneath cells as a fine coating on the substratum. In sparse cultures, Sp46 is detected in trails behind motile cells. In contrast, secreted fibronectin coalesces into fibers, most of which remain in contact with and on top of the cells; thus Sp46 does not preferentially bind to fibronectin. About 6 kD of the mass of human Sp46 is N-linked oligosaccharide, which is terminally sialated before secretion. Sp46 has a low glycine content, indicating that it is not a collagenlike protein. Its NH2-terminal sequence over the first 40 amino acids does not resemble any protein for which sequence information is available. Sp46 appears to be a novel extracellular glycoprotein, high-level constitutive expression of which is restricted to mesoderm-derived epithelial and endothelial cells. We therefore propose for it the name "mesosecrin." PDF | Back


Anchorage-independent growth of normal human mesothelial cells: a sensitive bioassay for EGF which discloses the absence of this factor in fetal calf serum.

La Rocca PJ, Rheinwald JG.

In Vitro Cell Dev Biol. 1985 Jan;21(1):67-72.

This laboratory recently reported that normal human mesothelial cells require epidermal growth factor (EGF) and hydrocortisone (HC), in addition to fetal calf serum and a complex defined medium component, in order to grow optimally in surface culture. We report here that this normal cell type also forms large colonies at high efficiency in semi-solid medium, but exhibits more stringent serum and EGF requirements for anchorage-independent than for surface growth. Mesothelial cells are unable to divide at all in semi-solid medium without added EGF or with less than 2% serum, whereas they grow slowly but progressively in surface culture under such conditions. In semi-solid medium containing 20% serum and HC, mesothelial cells are stimulated to divide by the addition of as little as 30 pg/ml purified EGF. Human urine or male mouse plasma could substitute for purified EGF, yielding growth commensurate with the levels of EGF in these biological fluids previously measured by others using radioreceptor and radioimmune assays. Thus growth of mesothelial cells in semi-solid medium can serve as a highly sensitive assay of EGF biological activity which is unaffected by the presence of serum proteins. In addition, our results demonstrate that fetal calf serum does not provide mitogenic levels of EGF to cultured cells, raising the question of the identity of plasma and serum mitogens. PDF | Back


Cellular X-ray repair parameters of early passage squamous cell carcinoma lines derived from patients with known responses to radiotherapy.

Weichselbaum R, Dahlberg W, Little JB, Ervin TJ, Miller D, Hellman S, Rheinwald JG.

Br J Cancer. 1984 May;49(5):595-601.

We have investigated X-ray survival parameters and repair of potentially lethal damage ( PLDR ) in ten early passage squamous cell carcinoma cell lines derived from patients who were biopsied before initiation of radiotherapy or after radiation therapy failure. Radiosensitivity (D0) ranged from 1.07 to 1.93 (Gy), extrapolation numbers (-n) from 1.17 to 2.14 and PLD recovery at 24 h from 1.4 to 20.3. Despite significant differences in these parameters amongst the cell lines, a firm correlation between radiocurability and any individual radiobiological parameter could not be established. Our data suggest that the mechanisms associated with radioresistance are complex and that any single radiobiological parameter may not predict clinical success or failure. PDF | Back


A monoclonal antibody recognizing a keratin filament protein in a subset of transitional and glandular epithelia.

Nadakavukaren KK, Summerhayes IC, Salcedo BF, Rheinwald JG, Chen LB.

Differentiation. 1984;27(3):209-20.

Monoclonal antibodies were raised against the detergent-insoluble cytoskeletal fraction of the human bladder carcinoma cell line, EJ. By immunofluorescence, one of these antibodies, H10-1, localized to keratin filaments in EJ and three out of five other transitional-cell carcinoma lines. Primary normal-human urothelial cells in culture were not recognized by H10-1. Strong staining of keratin filaments was also seen in 11 human adenocarcinoma cell lines, but it was only seen in a subpopulation of cells in one out of four squamous-cell carcinoma lines and not at all in two normal diploid human epidermal keratinocyte strains. Immunofluorescence on frozen sections of mouse, human, and rabbit tissues showed that H10-1 recognized the upper layer of the transitional epithelium of mouse and rabbit bladder, and a subset of simple epithelia (mouse stomach glands and some colon mucosal glands, but not kidney tubules, small intestine, ovary germinal epithelium, or endometrium; some human colon mucosal glands and glandular breast epithelium, but not endometrium). The antigen which is recognized by this antibody was not detected in examples of stratified squamous epithelium (mouse skin, nonglandular stomach, or esophagus; human skin, esophagus, or rectum) or nonepithelial tissue. On frozen sections of human tumors, the H10-1 antibody recognized 9 out of 14 colon carcinomas and 4 out of 6 breast carcinomas but did not recognize two sarcomas or a melanoma. This antibody apparently does not recognize its antigen in the presence of sodium dodecyl sulfate (SDS) because brief exposure of methanol-fixed cells to 0.1% SDS reversibly inhibited the binding of the H10-1 monoclonal antibody to keratin filaments by immunofluorescence, even when the binding of a rabbit polyclonal antikeratin antibody was unaffected. Two-dimensional gel-electrophoretic analysis of keratin-enriched fractions which had been isolated from various cell lines disclosed a good correlation between the presence of a 47-kdalton, pI-5.35 polypeptide and positive immunofluorescence with this antibody. These observations suggest that a certain keratin or keratin-associated protein is specifically expressed in a subpopulation of transitional and simple epithelial cells, and cultured cells derived from these epithelia. The monoclonal antibody described here may therefore be useful for increasing the precision of histological classification of epithelia, as well as the verification of the origin of certain cultured cell lines. PDF | Back


Coexpression of simple epithelial keratins and vimentin by human mesothelium and mesothelioma in vivo and in culture.

LaRocca PJ, Rheinwald JG.

Cancer Res. 1984 Jul;44(7):2991-9.

We have determined the intermediate filament proteins present in normal and malignant mesothelium in vivo. Pure sheets of normal lung mesothelium were obtained by transfer to agarcoated slides or by gentle scraping and cytocentrifugation. Cytoplasmic filament networks in the mesothelium were labeled via indirect immunofluorescence both by anti-Mr 40,000 keratin and anti-vimentin antisera. Two-dimensional gel electrophoresis of the Triton:high-salt-insoluble proteins of normal lung mesothelium disclosed the presence of vimentin and all but the largest (Mr 55,000) of the four simple epithelial keratins synthesized by mesothelial cells in culture. Samples of three peritoneal and three pleural mesotheliomas were found to contain either all four simple epithelial keratins or all but the Mr 55,000 keratin. Notably, none of the keratins characteristic of stratified and many glandular epithelia and their malignant forms was present in these mesotheliomas. Two mesothelioma samples from which the tumor cells could be obtained free of other cell types were found to contain vimentin as well as simple epithelial keratins and to synthesize these same proteins during short-term culture. None of the mesotheliomas placed in culture grew progressively in medium optimal for the growth of normal mesothelial cells. These data demonstrate that malignant mesothelial cells preserve the normal pattern of intermediate filament protein synthesis, including coexpression of simple epithelial keratins and vimentin, and suggest the use of this characteristic as an aid in the identification of cells of mesothelial origin. PDF | Back


Polycyclic aromatic hydrocarbon mutagenesis of human epidermal keratinocytes in culture.

Allen-Hoffmann BL, Rheinwald JG.

Proc Natl Acad Sci U S A. 1984 Dec;81(24):7802-6.

We have developed a culture system for detecting and isolating rare hypoxanthine phosphoribosyltransferase-deficient mutants of human epidermal keratinocytes. A thioguanine-resistant variant, 3T3M1, of the Swiss mouse fibroblast line 3T3 was used as a feeder layer to support clonal growth of mutant keratinocytes. A near-diploid, epidermal squamous cell carcinoma line, SCC-13Y, was used as a prototype to determine mutagen treatment conditions, plating density, and phenotypic expression time for maximum mutant recovery. To extend this system to normal keratinocytes, we improved the culture conditions by adding insulin, adenine, and Ham's nutrient mixture F-12, which increased colony-forming efficiencies to 30% in early passage and made feasible the detection of rare mutants in normal epidermal keratinocyte populations. We have quantitated mutation in SCC-13Y and three strains of normal human epidermal keratinocytes after exposure to polycyclic aromatic hydrocarbons, which are activated to their mutagenic forms by cellular mixed-function oxidases. 7,12-Dimethylbenz[a]anthracene and benzo[a]pyrene caused almost no cytotoxicity, but induced thioguanine-resistant mutants at frequencies as much as 50-fold higher than the spontaneous frequency of approximately 10(-6). The mutants were aminopterin-sensitive and possessed no measurable hypoxanthine phosphoribosyltransferase activity; their behavior was indistinguishable from that of keratinocytes cultured from individuals with Lesch-Nyhan syndrome. This mutagenesis assay system should also be applicable to other feeder layer-dependent human epithelial cell types, such as urothelial, mammary, and tracheal epithelial cells. PDF | Back


Tissue specificity of epithelial keratins: differential expression of mRNAs from two multigene families.

Kim KH, Rheinwald JG, Fuchs EV.

Mol Cell Biol. 1983 Apr;3(4):495-502.

Human epithelial cells cultured from stratified and simple squamous tissues all produce keratins of 40,000 to 58,000 daltons, but within this range the number and sizes vary with different epithelial cells. We have shown that this tissue-specific variation in the keratins is not due to posttranslational modification or processing, but rather to the differential expression of a family of heterogeneous but closely related mRNAs. All of these epithelial keratin mRNAs can be further grouped into two distinct subfamilies by their ability to hybridize with either of two cloned epidermal keratin cDNAs. All of the keratin mRNAs hybridize to one or the other, but not both, of the two cloned cDNAs. However, the mRNAs within each group hybridize with varying degrees of stringency, indicating that they are of similar but not identical sequence. Both types of keratin mRNAs are always expressed in every epithelial cell line studied, suggesting that filament assembly is dependent on the presence of both types of keratins. Within each of these two groups, the slight sequence differences in each class may reflect subtle tissue-specific variations in the structural and functional requirements of the epithelial cytoskeleton. PDF | Back


Regulation of the cytoskeleton in mesothelial cells: reversible loss of keratin and increase in vimentin during rapid growth in culture.

Connell ND, Rheinwald JG.

Cell. 1983 Aug;34(1):245-53.

Human mesothelial cells grew rapidly in culture when provided with serum, EGF, and hydrocortisone, adopting a fibroblastoid shape and forming parallel, multilayered arrays at saturation density. In the absence of EGF, the cells grew slowly to a flat, epithelioid monolayer similar to their normal pattern in vivo. Mesothelial cells normally have a high keratin and a low vimentin content in vivo. In culture, rapidly growing cells greatly reduced synthesis and content of their four major keratins to levels undetectable by immunofluorescence in most cells, but keratin synthesis and content returned to high levels whenever growth slowed. Vimentin synthesis and content was high during serial culture, but decreased several-fold in nondividing cells. The unique ability of the mesothelial cell to reversibly alter its morphology and intermediate filament composition is of unknown function and mechanism, but accounts for the morphological heterogeneity and the presence of keratin-negative cells in mesotheliomas. PDF | Back


The mesothelial keratins: a new family of cytoskeletal proteins identified in cultured mesothelial cells and nonkeratinizing epithelia.

Wu YJ, Parker LM, Binder NE, Beckett MA, Sinard JH, Griffiths CT, Rheinwald JG.

Cell. 1982 Dec;31(3 Pt 2):693-703.

The cytoskeletal proteins of cultured normal human mesothelial cells were found to consist of six major components, including actin, vimentin, the 40 kd keratin and the 44, 52 and 55 kd proteins, plus a minor 46 kd protein. Two-dimensional gel electrophoresis, peptide mapping and immunoprecipitation tests showed that the 40-55 kd mesothelial proteins are a family of keratins distinct in size, charge or peptide map from the "epidermal keratins" synthesized by cultured keratinocytes. Unique combinations of keratins from the epidermal and mesothelial keratin families were found to be synthesized by cultured bladder, esophageal, conjunctival, mammary, exocervical and ovarian surface epithelial cells. Mesothelial cells were the only epithelial cell type that synthesized vimentin at more than trace levels. We have also found that many carcinoma cell lines express keratins different from those of their cell type of origin. PDF | Back


Human squamous cell carcinoma in culture: a defect in terminal differentiation and its relation to malignancy.

Rheinwald JG.

Natl Cancer Inst Monogr. 1982;60:133-8.

Thirteen primary squamous cell carcinomas of the epidermis and of the oral and pharyngeal epithelium were cultured with a 3T3 fibroblast feeder layer, a system originally developed for clonal growth and long-term serial cultivation of normal human keratinocytes. Six of these tumors could be propagated indefinitely as established cell lines. They formed rapidly growing well-differentiated squamous cell carcinomas when injected sc into athymic (nude) mice. The squamous cell carcinoma lines possessed different aneuploid karyotypes. They displayed subtle differences in colony morphology such that the were visually distinguishable from one another as well as from normal keratinocytes. The lines also varied greatly in their dependence on the fibroblast feeder layer for clonal growth in surface culture. Only 1 line could form large colonies with high efficiency in semisolid medium; the others grew only abortively under this condition and eventually differentiated terminally to form cornified envelopes. Progressive growth in semisolid medium, therefore, was not a useful in vitro marker of malignant transformation for these cancer cells of keratinocyte origin. However, a property shared by all the squamous cell carcinoma lines was a subnormal rate of commitment to terminal differentiation during incubation in suspension culture. Deprivation of anchorage triggers commitment so rapidly in normal keratinocyte populations that no cell remain viable after 2 days in semisolid medium. In contrast, after this same-period, all 6 lines retained more than 20% of their original colony-forming ability when replated in surface culture. This phenotype of increased survival capacity in semisolid medium promises to be a useful selective marker for the detection of rare malignant keratinocytes within large normal keratinocyte populations. PDF | Back


A new small (40 kd) keratin filament protein made by some cultured human squamous cell carcinomas.

Wu YJ, Rheinwald JG.

Cell. 1981 Sep;25(3):627-35.

We examined the keratin proteins of nine cell lines cultured from human squamous cell carcinomas (SCCs) of the epidermis and oral epithelium.  All of these lines contained the four major keratins (46 kd, 50 kd, 56 kd, and 58 kd) found in normal keratinocytes cultured from these epithelia.  A 40 kd protein was also present in the Triton-insoluble cytoskeletal fraction of three of the lines.  The 40 kd protein had an isoelectric point of 5.3, near that of the 46 kd and 50 kd keratins.  It was precipitated by an antiserum against total s. corneum keratins and also by an antiserum against purified 46 kd keratin.  An antiserum raised against the purified 40 kd protein had a high titer against the 40 kd protein, detected by immunoprecipitation, and did not cross-react with the 46-58 kd keratins.  The anti-40 kd serum stained, by indirect immunofluorescence, a cytoplasmic filament network in cells of the SCC lines that synthesize the 40 kd protein.  These filaments were of a pattern indistinguishable from that stained by antiserum against total s. corneum keratins, and the pattern was not altered by treatment of the cells with Colcemid.  The anti-40 kd serum did not stain the SCC lines that do not synthesize the 40 kd protein, normal human newborn foreskin keratinocytes or fibroblast, or 3T3, HeLa, or XB cells. Keratinocytes cultured from uninvolved epidermal or oral tissue of patients whose tumors had given rise to SCC lines that synthesize the 40 kd protein were also not stained by the anti-40 kd serum.  Synthesis of the 40 kd protein was greatly reduced in tumors formed by the SCC lines that make this protein in culture.  Partial proteolytic digestion maps of the 40 kd protein were unique, but very similar to those of the 46-59 kd keratins. These results constitute strong evidence that the 40 kd protein is a keratin and is neither a breakdown product nor an incomplete translation product of a gene for one of the later keratins.  it is the smallest epidermal keratin yet to be described.  The 40 kd keratin may be the product of a normally unexpressed gene or transcript that often becomes activated during the course of keratinocyte transformation.  Like the larger keratins, the level of its expression is regulated and is different in cells growing in vivo and in culture. PDF | Back


Tumorigenic keratinocyte lines requiring anchorage and fibroblast support cultures from human squamous cell carcinomas.

Rheinwald JG, Beckett MA.

Cancer Res. 1981 May;41(5):1657-63.

We have established cell lines from six human squamous cell carcinomas (SCC) of the epidermis and tongue, using culture methods previously developed for clonal growth and serial cultivation of normal keratinocytes. The SCC lines all form rapidly growing, well-differentiated SCC's or progressively growing squamous cysts in nude mice. In contrast to normal keratinocytes, SCC cells form unstratified or very poorly stratifying colonies and do not require epidermal growth factor for sustained growth. The SCC lines vary in their requirement for a fibroblast feeder layer to support clonal growth, as normal keratinocytes possess. Only one line forms large, progressively growing colonies at high efficiency in semisolid medium; the other five lines exhibit only a small amount of abortive growth in semisolid medium, after which the cells appear to rapidly degenerate. These results demonstrate that SCC's often grow as established lines in culture, but they frequently possess in vitro growth requirements similar to those of normal keratinocytes. Consequently, neither semisolid medium nor standard surface culture media are appropriate for initiating primary SCC cultures or for selecting transformants out of carcinogen-treated keratinocyte populations, because they do not provide conditions permissive for the growth of many malignant keratinocytes. PDF | Back


Failure of hydrocortisone or growth factors to influence the senescence of fibroblasts in a new culture system for assessing replicative lifespan.

Didinsky JB, Rheinwald JG.

J Cell Physiol. 1981 Oct;109(1):171-9.

It has been reported that the replicative lifespan of human fibroblasts can be substantially extended by supplementing the growth medium with hydrocortisone or increased levels of serum proteins. These observations have been made only on cell populations transferred many times at high cell density, and cumulative population doublings have been recorded, rather than a more direct measure of cell division potential. We have measured the replicative potential of human fibroblasts cultured so as to avoid conditions of high cell density, medium depletion, and departure from exponential growth. Two fetal lung and two newborn foreskin fibroblast strains were serially passaged in the presence or absence of hydrocortisone (HC), epidermal growth factor (EGF), and fibroblast growth factor (FGF) until they senesced. At each passage cells were plated at densities sufficiently low that colony-forming efficiency could be calculated. We determined cumulative population doublings and also estimated the number of cell generations attained under each condition. FGF caused small but possibly significant changes, while HC and EGF failed to substantially alter replicative lifespan. The reported effect of HC on the doubling potential of fetal lung fibroblasts is therefore not an inevitable action of this hormone on the senescence mechanism, but may instead depend for its apparent activity on the passage regimen used. The fibroblast's insensitivity to EGF as a modulator of replicative potential, as compared with the keratinocyte, whose lifespan can be tripled by EGF, implies that the mechanisms limiting the replicative potential of these two cell types are not identical. PDF | Back


Defective terminal differentiation in culture as a consistent and selectable character of malignant human keratinocytes.

Rheinwald JG, Beckett MA.

Cell. 1980 Nov;22(2 Pt 2):629-32.

Culture conditions can be manipulated to vary the rate at which keratinocytes (stratified squamous epithelial cells) become committed to differentiate terminally. When deprived of anchorage in semisolid medium, normal human keratinocytes irreversibly lose the ability to reinitiate growth in surface culture with a t1/2 of 3 hr and then proceed to form cornified envelopes. We examined six established lines from human squamous cell carcinomas (SCCs) for defects in this function. One SCC line which grew progressively in semi-solid medium could not be induced to form cornified envelopes. The other five lines, which grew abortively, at best, in semisolid medium, formed envelopes under this condition but at subnormal rates. During anchorage deprivation the SCC lines became committed to differentiate much more slowly than normal, with t1/2's of 24--144 hr. SCC cells therefore possess at least a partial defect in the triggering of terminal differentiation. In vivo, such an alteration may permit malignant behavior by evading an important tissue-specific mechanism for limiting growth. In culture, the phenotype of increased survival in semi-solid medium may be used to detect and select malignantly transformed keratinocytes. PDF | Back


Epidermal growth factor and the multiplication of cultured human epidermal keratinocytes.

Rheinwald JG, Green H.

Nature. 1977 Feb 3;265(5593):421-4.

The culture lifetime of epidermal cells of newborn humans is increased from 50 to 150 generations by adding to the medium epidermal growth factor, a polypeptide mitogen. EGF seems to delay senescence of the cells by maintaining them in a state further removed from terminal differentiation. This effect is revealed by a greater ability of the cells to survive subculture and initiate new colonies, but not necessarily by an increased growth rate. PDF | Back


Properties of an epithelial cell type in culture: the epidermal keratinocyte and its dependence on products of the fibroblast.

Green H, Rheinwald JG, Sun TT.

Prog Clin Biol Res. 1977;17:493-500.

Keratinocytes of stratified squamous epithelium can be grown serially in culture and retain the various markers typical of their form of differentiation. In order to form colonies at each transfer, the keratinocytes must be suitably supported by fibroblasts. Established keratinocyte lines of teratomal origin show this dependence, as do diploid strains of finite culture life derived from human skin. For at least some keratinocyte lines, this requirement can be satisfied by soluble products elaborated by the fibroblasts. It is suggested that epithelial cells in general may not be independent cell types and that their poor cultivability may be due to failure to provide suitable fibroblast support. The existence of a number of established lines of epithelial origin that can grow without such support and of lines of fibroblastic origin which cannot support keratinocytes suggests that both epithelial dependence and the fibroblast supporting function can sometimes be lost in established cell lines. Back


Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells.

Rheinwald JG, Green H.

Cell. 1975 Nov;6(3):331-43.

Human diploid epidermis epidermal cells have been successfully grown in serial culture. To initiate colony formation, they require the presence of fibroblasts, but proliferation of fibroblasts must be controlled so that the epidermal cell population is not overgrown. Both conditions can be achieved by the use of lethally irradiated 3T3 cells at the correct density. When trypsinized human skin cells are plated together with the 3T3 cells, the growth of the human fibroblasts is largely suppressed, but epidermal cells grow from single cells into colonies. Each colony consists of keratinocytes ultimately forming a stratified squamous epithelium in which the dividing cells are confined to the lowest layer(s). Hydrocortisone is added to the medium, since in secondary and subsequent subcultures it makes the colony morphology more oderly and distinctive, and maintains proliferation at a slightly greater rate. Under these culture conditions, it is possible to isolate keratinocyte clones free of viable fibroblasts. Like human diploid fibroblasts, human diploid keratinocytes appear to have a finite culture lifetime. For 7 strains studied, the culture lifetime ranged from 20-50 cell generations. The plating efficiency of the epidermal cells taken directly from skin was usually 0.1-1.0%. On subsequent transfer of the cultures initiated from newborns, the plating efficiency rose to 10% or higher, but was most often in the range of 1-5% and dropped sharply toward the end of their culture life. The plating efficiency and culture lifetime were lower for keratinocytes of older persons. PDF | Back


Formation of a keratinizing epithelium in culture by a cloned cell line derived from a teratoma.

Rheinwald JG, Green H.

Cell. 1975 Nov;6(3):317-30.

From a transplantable mouse teratoma it has been possible to derive an established keratinizing cell line (XB) which grows well in cultures containing lethally irradiated 3T3 fibroblasts at the correct density. Single cells of the keratinizing line grow into colonies each consisting of a stratified squamous epithelium. The keratinizing nature of the colonies has been demonstrated by specific staining with Rhodanile blue, and by light and electron microscopy of sections through the colonies. A function of fibroblasts appears to be a strict requirement for keratinization and an important though less strict requirement for cell growth. The fibroblast function can be carried out by medium harvested from 3T3 cultures. It is possible to detect keratinizing colonies in primary cultures of disaggregated teratoma cells combined with 3T3 cells. Such colonies appeared in cultures of a transplantable teratoma with an overall frequency of 6 X 10(-6) of the cells plated. Nonkeratinizing colonies of cells with otherwise very similar appearance were about 10 fold more abundant. Since both the keratinizing and the related nonkeratinizing colonies can be identified in the living state, it is possible to isolate them from the primary cultures. PDF | Back


Growth of cultured mammalian cells on secondary glucose sources.

Rheinwald JG, Green H.

Cell. 1974 Aug;2(4):287-93.

Mammalian cells can grow in culture at very low glucose concentrations. They can also grow using starch or maltose as a secondary source of glucose if hydrolytic enzymes (amylase and/or maltase) are available to release the glucose. The serum supplement in the culture medium provides these enzymes in amount adequate to permit growth at as rapid rate as when free glucose is added. Owing to the relatively slow liberation of glucose from the secondary sources, the cells produce less lactic acid, and the culture medium does not become acidic.

If the amount of hydrolytic enzyme in the serum supplement is reduced by heat inactivation, the rate of glucose liberation Is further reduced. As a result, glucose continues to be released into the medium even at high cell densities, when all glucose added directly to control cultures has been consumed at a time. For this reason, the cells survive longer at a high density on secondary glucose sources than on free glucose. Use of such a culture system should have important practical advantages in maintaining dense cultures of any mammalian cell type.

Medium containing secondary glucose sources and serum whose hydrolytic enzymes have been completely inactivated should be a selective medium for the corresponding cellular enzymes. Attempts to select for cell lines able to grow using their own amylase or maltase were not successful, but calculations based on embryonic pancreatic cells, knows to synthesize amylase, showed that the amount of enzyme required should be quite low in comparison with that present in the differentiated sate. The possibilities of selection for a differentiated function in cell culture have been very little explored, and such an approach may be fruitful if applied to the right cell types. PDF | Back


A transmissible plasmid controlling camphor oxidation in Pseudomonas putida.

Rheinwald JG, Chakrabarty AM, Gunsalus IC.

Proc Natl Acad Sci U S A. 1973 Mar;70(3):885-9.

Earlier papers demonstrated an extensive genetic exchange among fluorescent Pseudomonads; this one documents for genes specifying enzymes of peripheral dissimilation an extrachromosomal array, segregation, and frequent interstrain transfer. A hypothesis is presented of a general mechanism for the formation and maintenance of metabolic diversity. The example used, the path of oxidative cleavage of the carbocyclic rings of the bicyclic monoterpene D- and L-camphor, terminates in acetate release and isobutyrate chain debranching. By transduction, two gene linkage groups are shown for the reactions before and after isobutyrate. The group for reactions before isobutyrate is plasmid borne, cotransferable by conjugation, mitomycin curable, and shows a higher segregation rate from cells that are multiplasmid rather then carrying a single plasmid. The genes that code for isobutyrate and essential anaplerotic and amphibolic metabolism are chromosomal. By conjugation plasmid-borne genes are transferred at a higher frequency than are chromosomal, and are transferred in homologous species. Most isobutyrate-positive fluorescent pseudomonad strains will accept and express the camphor plasmid. PDF | Back

© 2010 James Rheinwald