Murine Teratocarcinoma Stem Cells Research Articles

X inactivation: a histone protects from reprogramming by the frog

Have you seen?15 June 2011free access X inactivation: a histone protects from reprogramming by the frog Anton Wutz Corresponding Author Anton Wutz Department of Biochemistry, Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Cambridge, UK Search for more papers by this author Anton Wutz Corresponding Author Anton Wutz Department of Biochemistry, Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Cambridge, UK Search for more papers by this author Author Information Anton Wutz 1 1Department of Biochemistry, Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Cambridge, UK *Correspondence to: [email protected] The EMBO Journal (2011)30:2310-2311https://doi.org/10.1038/emboj.2011.172 There is an Article (June 2011) associated with this Have you seen?. PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Genes on the inactive X chromosome (Xi) of female mammals are repressed in a remarkably stable manner and reactivation of transcription is generally not observed unless the cell is reprogrammed to an early embryonic type. In this issue of The EMBO Journal, Pasque et al (2011) use a reprogramming system in frog oocytes to study the stability of Xi chromatin in cells of different developmental stages and identify the histone variant macroH2A as a factor preventing transcriptional activation. X inactivation provides dosage compensation between the sexes in mammals. One of the two X chromosomes is transcriptionally silent in female somatic cells and as a consequence a single X chromosome is active in males and females. In somatic cells, several mechanisms including DNA methylation and histone modifications are thought to contribute to maintaining silencing. As a result, genes on the Xi cannot be reactivated efficiently by interfering with epigenetic mechanisms. This contrasts the situation in embryogenesis. X inactivation is initiated in early female embryos by the non-coding Xist RNA. Pluripotent female mouse embryonic stem cells (ESCs) possess two active X chromosomes and initiate X inactivation upon entry into differentiation. Mouse epiblast-derived stem cells (EpiSCs) are also pluripotent but correspond to a stage when X inactivation has already been initiated. In later development gene repression on the Xi becomes progressively stabilized finally resulting in an irreversibly silenced chromosome. The stability of gene repression on the Xi appears to reflect the differentiation state of a cell and in the case of ESCs marks their developmental plasticity. Consistent with this view is the observation that cell fusion of somatic female cells with mouse ESCs results in reactivation of the Xi (Takagi et al, 1983). Xi reactivation is also associated with reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) in mice (Maherali et al, 2007). Xi reactivation thereby overlaps with the establishment of pluripotency. The mechanism for Xi reactivation has, therefore, attracted attention for its implications for understanding reprogramming. In this issue, Pasque et al (2011) introduce a new system for probing the stability of the Xi. Nuclear transfer into Xenopus oocytes is used to show that the Xi from female mouse embryonic fibroblasts (MEFs) remains repressed in frog oocytes. However, the Xi from differentiating ESCs and EpiSCs is reactivated (see Figure 1). Comparison of the chromatin composition of the Xi between the different donor cell types identifies the histone variant macroH2A as a key determinant of Xi stability. MacroH2A is present on the Xi in somatic cells but not in ESCs or EpiSCs. In addition, depletion of macroH2A from the somatic donor cells before nuclear transfer leads to not only elevated reactivation of the Xi but also reactivation of other genes associated with pluripotency including the transcription factors Oct4 and Sox2. This indicates a wider role of macroH2A in maintaining transcriptional repression of silent genes in somatic cells. Intriguingly, this study finds that other chromatin marks such as DNA methylation or histone methylation do not interfere with the reactivation of the Xi from EpiSCs. Figure 1.Reactivation of the Xi by nuclear transfer into frog oocytes. Nuclei from female mouse ESCs, EpiSCs and MEFs are transplanted into the reprogramming environment of frog oocytes. The activity of genes on the Xi was then analysed to assess reactivation of transcription. Whereas the Xi from differentiated ESCs and EpiSCs was reactivated the Xi derived from MEFs remained silent. Lack of reactivation correlated with the presence of mcaroH2A on the Xi in MEFs, which was maintained on the Xi after nuclear transfer. Download figure Download PowerPoint MacroH2A has an unusual structure for a histone with a large non-histone domain added to its C-terminus which has been implicated in blocking transcription and as a binding domain (Takahashi et al, 2002; Karras et al, 2005). It was first discovered as a component of the Xi heterochromatin by its specific enrichment on the Xi in somatic cells (Costanzi and Pehrson, 1998). Previous work in MEFs has implicated macroH2A in Xi stability (Hernandez-Munoz et al, 2005). However, mutation of the macroH2A1 gene in mice is compatible with female development, suggesting that macroH2A1 contributes to stabilizing the Xi together with other mechanisms (Changolkar et al, 2007). Also, in MEFs macroH2A enrichment on the Xi appears to require Xist (Csankovszki et al, 1999). The fact that Xist was lost from the Xi after nuclear transfer into frog oocytes but macroH2A was maintained indicates differences between the frog and mouse systems. One important aspect is the absence of cell division during reprogramming in frog oocytes. Using appropriate controls early effects of the frog reprogramming environment on chromatin can be observed. Regulation of macroH2A incorporation has recently been studied in mouse development. In particular, it has been shown that macroH2A is displaced from chromatin after fertilization, suggesting that exclusion of macroH2A from chromatin is associated with a period of genome-wide reprogramming in pre-implantation development (Nashun et al, 2011). Moreover, exchange of critical amino acids causing incorporation of macroH2A into chromatin in early embryos is detrimental for development. It is, therefore, likely that histone H2A variants have a major role in determining chromatin plasticity and developmental potential. Importantly, macroH2A might have a similar role in restricting gene expression for preventing tumourigenesis. A recent study has demonstrated that in aggressive melanoma loss of macroH2A expression correlates with the activation of known oncogenes (Kapoor et al, 2011). Insights into the Xi facultative heterochromatin obtained in the study by Pasque et al (2011) have, therefore, implications for understanding iPSC reprogramming and in tumourigenesis. Conflict of Interest The author declares that he has no conflict of interest. References Changolkar LN, Costanzi C, Leu NA, Chen D, McLaughlin KJ, Pehrson JR (2007) Developmental changes in histone macroH2A1-mediated gene regulation. Mol Cell Biol 27: 2758–2764CrossrefCASPubMedWeb of Science®Google Scholar Costanzi C, Pehrson JR (1998) Histone macroH2A1 is concentrated in the inactive X chromosome of female mammals. Nature 393: 599–601CrossrefCASPubMedWeb of Science®Google Scholar Csankovszki G, Panning B, Bates B, Pehrson JR, Jaenisch R (1999) Conditional deletion of Xist disrupts histone macroH2A localization but not maintenance of X inactivation. Nat Genet 22: 323–324CrossrefCASPubMedWeb of Science®Google Scholar Hernandez-Munoz I, Lund AH, van der Stoop P, Boutsma E, Muijrers I, Verhoeven E, Nusinow DA, Panning B, Marahrens Y, van Lohuizen M (2005) Stable X chromosome inactivation involves the PRC1 Polycomb complex and requires histone MACROH2A1 and the CULLIN3/SPOP ubiquitin E3 ligase. Proc Natl Acad Sci USA 102: 7635–7640CrossrefCASPubMedWeb of Science®Google Scholar Kapoor A, Goldberg MS, Cumberland LK, Ratnakumar K, Segura MF, Emanuel PO, Menendez S, Vardabasso C, Leroy G, Vidal CI, Polsky D, Osman I, Garcia BA, Hernando E, Bernstein E (2011) The histone variant macroH2A suppresses melanoma progression through regulation of CDK8. Nature 468: 1105–1109CrossrefCASWeb of Science®Google Scholar Karras GI, Kustatscher G, Buhecha HR, Allen MD, Pugieux C, Sait F, Bycroft M, Ladurner AG (2005) The macro domain is an ADP-ribose binding module. EMBO J 24: 1911–1920Wiley Online LibraryCASPubMedWeb of Science®Google Scholar Maherali N, Sridharan R, Xie W, Utikal J, Eminli S, Arnold K, Stadtfeld M, Yachechko R, Tchieu J, Jaenisch R, Plath K, Hochedlinger K (2007) Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell Stem Cell 1: 55–70CrossrefCASPubMedWeb of Science®Google Scholar Nashun B, Yukawa M, Liu H, Akiyama T, Aoki F (2011) Changes in the nuclear deposition of histone H2A variants during pre-implantation development in mice. Development (Cambridge, England) 137: 3785–3794CrossrefCASWeb of Science®Google Scholar Pasque V, Gillich A, Garrett N, Gurdon JB (2011) Histone variant macroH2A confers resistance to nuclear reprogramming. EMBO J 30: 2373–2387Wiley Online LibraryCASPubMedWeb of Science®Google Scholar Takagi N, Yoshida MA, Sugawara O, Sasaki M (1983) Reversal of X-inactivation in female mouse somatic cells hybridized with murine teratocarcinoma stem cells in vitro. Cell 34: 1053–1062CrossrefCASPubMedWeb of Science®Google Scholar Takahashi I, Kameoka Y, Hashimoto K (2002) MacroH2A1.2 binds the nuclear protein Spop. Biochimica et biophysica acta 1591: 63–68CrossrefCASPubMedWeb of Science®Google Scholar Previous ArticleNext Article Read MoreAbout the coverClose modalView large imageVolume 30,Issue 12,June 15, 2011“The Will-o’-the-Wisp” - Macro photograph of an Albizia flower, captured by Stéphane Noselli of the Institute of Developmental Biology and Cancer (IBDC), University of Nice-Sophia Antipolis. Volume 30Issue 1215 June 2011In this issue FiguresReferencesRelatedDetailsLoading ...

Expression of REX-1, a Gene Containing Zinc Finger Motifs, Is Rapidly Reduced by Retinoic Acid in F9 Teratocarcinoma Cells

In the presence of retinoic acid (RA), cultured F9 murine teratocarcinoma stem cells differentiate into nontumorigenic cells resembling the extraembryonic endoderm of the early mouse embryo. By differential hybridization screening of an F9 cell cDNA library, we isolated a 1,745-nucleotide cDNA for a gene, REX-1 (for reduced expression), whose steady-state mRNA level began to decline in F9 cells in monolayer culture within 12 h after the addition of RA. By 48 to 96 h after RA treatment of F9 cells in monolayer culture, the REX-1 steady-state mRNA level was more than sevenfold lower than the level in undifferentiated F9 stem cells. The REX-1 mRNA decrease did not result from the reduction in cell growth rate associated with the differentiation process, since the REX-1 mRNA level did not decline in F9 cells that were partially growth arrested after 48 h of isoleucine deprivation. The RA-associated REX-1 mRNA decrease resulted primarily from a reduction in the transcription rate of the REX-1 gene in the presence of RA. In contrast to results in F9 cells, we have been unable thus far to detect REX-1 mRNA in day 7.5 to 12.5 mouse embryo RNA samples or in the P19 teratocarcinoma stem cell line. The putative REX-1 protein identified by DNA sequence analysis contains four repeats of the zinc finger nucleic acid-binding motif and a potential acidic activator domain, suggesting that REX-1 encodes a regulatory protein. The REX-1 gene is not identical to the previously reported murine genes that encode zinc finger-containing proteins.

Expression of REX-1, a gene containing zinc finger motifs, is rapidly reduced by retinoic acid in F9 teratocarcinoma cells.

In the presence of retinoic acid (RA), cultured F9 murine teratocarcinoma stem cells differentiate into nontumorigenic cells resembling the extraembryonic endoderm of the early mouse embryo. By differential hybridization screening of an F9 cell cDNA library, we isolated a 1,745-nucleotide cDNA for a gene, REX-1 (for reduced expression), whose steady-state mRNA level began to decline in F9 cells in monolayer culture within 12 h after the addition of RA. By 48 to 96 h after RA treatment of F9 cells in monolayer culture, the REX-1 steady-state mRNA level was more than sevenfold lower than the level in undifferentiated F9 stem cells. The REX-1 mRNA decrease did not result from the reduction in cell growth rate associated with the differentiation process, since the REX-1 mRNA level did not decline in F9 cells that were partially growth arrested after 48 h of isoleucine deprivation. The RA-associated REX-1 mRNA decrease resulted primarily from a reduction in the transcription rate of the REX-1 gene in the presence of RA. In contrast to results in F9 cells, we have been unable thus far to detect REX-1 mRNA in day 7.5 to 12.5 mouse embryo RNA samples or in the P19 teratocarcinoma stem cell line. The putative REX-1 protein identified by DNA sequence analysis contains four repeats of the zinc finger nucleic acid-binding motif and a potential acidic activator domain, suggesting that REX-1 encodes a regulatory protein. The REX-1 gene is not identical to the previously reported murine genes that encode zinc finger-containing proteins.

Spontaneous differentiation of murine teratocarcinoma stem cells at low temperature

Embryonal carcinoma cells differentiate in vitro after treatment with different drugs or under certain culture conditions. We show here that at 31 degrees C PCC-aza-1 cells differentiated spontaneously into cells with characteristics resembling those of epithelial cells. With this temperature, we observed morphological changes; reorganization of the cytoskeleton with polymerization of actin cables, tight junctions, and cytokeratin endo A messenger RNA and protein expression. The cells also became totally permissive to Py virus but remained resistant to SV40 infection. This new pathway of differentiation has the advantage of being reversible at 37 degrees C.

Isolation of cDNA clones for genes exhibiting reduced expression after differentiation of murine teratocarcinoma stem cells.

In the absence of retinoic acid, PSA-G teratocarcinoma stem cells spontaneously differentiate at a moderate frequency into fibroblast-like cells. In the presence of retinoic acid and dibutyryl cyclic AMP, PSA-G stem cells differentiate into parietal endoderm cells. We prepared a cDNA library from undifferentiated PSA-G teratocarcinoma stem cells; this cDNA library was then screened for gene sequences which exhibit a reduction in expression during the differentiation of these stem cells. From ca. 1,000 clones screened, eight independent sequences were isolated. The level of expression of these cloned genes decreases by 3.0-fold to more than 10-fold after differentiation of PSA-G cells into fibroblast-like cells. After treatment of either PSA-G or F9 teratocarcinoma cells with retinoic acid and dibutyryl cyclic AMP for 72 h, the expression of seven genes is inhibited by two- to fourfold. This decrease of clone-specific transcripts can be detected within 12 h after the addition of retinoic acid. Hybridization-selection and in vitro translation experiments identified the proteins encoded by three of the cloned genes: pST 6-23 codes for a 89,000-dalton protein, pST 7-105 codes for a 41,000-dalton protein, and pST 9-31 codes for a 34,000-dalton protein. The 89,000-dalton protein encoded by pST 6-23 is a heat shock protein. In vitro transcription experiments demonstrate that the retinoic acid-mediated decrease in pST 6-135- and pST 1-68-specific RNA occurs at the transcriptional level and that dibutyryl cyclic AMP acts posttranscriptionally to further depress the levels of these RNAs.

Isolation of cDNA clones for genes exhibiting reduced expression after differentiation of murine teratocarcinoma stem cells.

In the absence of retinoic acid, PSA-G teratocarcinoma stem cells spontaneously differentiate at a moderate frequency into fibroblast-like cells. In the presence of retinoic acid and dibutyryl cyclic AMP, PSA-G stem cells differentiate into parietal endoderm cells. We prepared a cDNA library from undifferentiated PSA-G teratocarcinoma stem cells; this cDNA library was then screened for gene sequences which exhibit a reduction in expression during the differentiation of these stem cells. From ca. 1,000 clones screened, eight independent sequences were isolated. The level of expression of these cloned genes decreases by 3.0-fold to more than 10-fold after differentiation of PSA-G cells into fibroblast-like cells. After treatment of either PSA-G or F9 teratocarcinoma cells with retinoic acid and dibutyryl cyclic AMP for 72 h, the expression of seven genes is inhibited by two- to fourfold. This decrease of clone-specific transcripts can be detected within 12 h after the addition of retinoic acid. Hybridization-selection and in vitro translation experiments identified the proteins encoded by three of the cloned genes: pST 6-23 codes for a 89,000-dalton protein, pST 7-105 codes for a 41,000-dalton protein, and pST 9-31 codes for a 34,000-dalton protein. The 89,000-dalton protein encoded by pST 6-23 is a heat shock protein. In vitro transcription experiments demonstrate that the retinoic acid-mediated decrease in pST 6-135- and pST 1-68-specific RNA occurs at the transcriptional level and that dibutyryl cyclic AMP acts posttranscriptionally to further depress the levels of these RNAs.

A monoclonal antibody which detects a 125 kd glycoprotein on embryonal carcinoma cells and is mitogenic for murine spleen cells

The isolation and characterisation of a monoclonal antibody (2H9), selected for reactivity against murine teratocarcinoma stem cells, is described. This antibody recognizes one major glycoprotein on the surface of embryonal carcinoma (EC) cells but detects multiple glycoprotein species on various murine or human derived cell types. The binding of the antibody to cells is abolished by treatment with periodate or neuraminidase. These results suggest that 2H9 antibody detects a carbohydrate determinant: this is apparently distinct from other anti-EC reactive monoclonal antibodies and a variety of lectins. These antibodies are mitogenic for a population of lymphocytes in murine spleen. The antibodies recognize numerous cell surface glycoproteins on the surface of the spleen cells.

Reversal of X-inactivation in female mouse somatic cells hybridized with murine teratocarcinoma stem cells in vitro

A series of near-diploid embryonal carcinoma-like hybrid cells were obtained from polyethylene glycolmediated cell fusion between murine embryonal carcinoma cells (PSA-6TG1 or OTF9-63) having one X chromosome and thymocytes or bone marrow cells from female mice carrying Cattanach's or Searle's translocation. Prior to fusion with EC cells the somatic cells are presumed to contain only one active X chromsome. Following hybrid formation, the chronology of X chromosome replication and the expression of X-linked gene Pgk-1 indicated that all X chromosomes contributed by both parents were active in these hybrids. Experiments were performed to rule out the possibility that the hybrids were formed by fusion of EC cells with rare somatic cells in which both X chromosomes were active. Taken together the data indicate that within four days of fusion there is reactivation of the entire inactive X chromosome.

Monoclonal antibody to murine embryos defines a stage-specific embryonic antigen expressed on mouse embryos and human teratocarcinoma cells.

A murine stage-specific embryonic antigen (SSEA3) is defined by reactivity with a monoclonal antibody prepared by immunization of a rat with 4- to 8-cell-stage mouse embryos. This antigenic determinant, present on oocytes, becomes restricted first to the inner cell mass at the blastocyst stage, and later to the primitive endoderm. Murine teratocarcinoma stem cells do not react with this antibody, whereas human teratocarcinoma stem cells are SSEA3-positive. This antigenic determinant is not expressed on a variety of other human and murine cell lines, but is found on the surface of human erythrocytes. It is a carbohydrate and is present on both cell-surface glycolipids and glycopeptides. These results demonstrate the feasibility of identifying stage-specific antigenic determinants with monoclonal antibody prepared against embryos. The need for thorough screening on a variety of cell types to establish developmentally important cross-reactivities is also emphasized.

Control of expression of histocompatibility antigens (H-2) and beta 2-microglobulin in F9 teratocarcinoma stem cells.

Murine teratocarcinoma stem cells, unlike most other cell types, do not express major histocompatibility antigens. The steady-state levels of beta 2-microglobulin and H-2 mRNA from F9-derived teratocarcinoma stem and differentiated cells were examined by blot hybridization using cloned DNA probes specific for these mRNAs. No H-2- or beta 2-microglobulin-specific RNA was detected in F9 teratocarcinoma stem cells (clone 12-1); thus, F9 teratocarcinoma stem cells (clone 12-1) contain no more than 1/10 the H-2 and beta 2-microglobulin mRNAs of the differentiated daughter cells (clone 12-1a). We suggest that this regulation of major histocompatibility antigen expression is due to transcriptional control of the major histocompatibility antigen genes, H-2 and beta 2-microglobulin. The transcriptional regulation of these genes is accompanied by a change in their DNase I sensitivity. Normally, transcriptionally inactive genes are DNase I resistant, while active genes are DNase I sensitive. In contrast, the silent major histocompatibility antigen genes of teratocarcinoma stem cells are more DNase I sensitive than the active genes of the differentiated cells.

Murine embryonal carcinoma cells: Universal targets for mammalian nk cells?

Natural killer (NK) cells from mice lyse murine teratocarcinoma stem cells in accordance with their appropriate cellular, genetic and interferon inducible features. In addition, rat and human putative NK cells were found to exhibit cytotoxicity of these embryonal carcinoma stem cells. By contrast, endodermal cells derived from such stem cells, either as cell lines or by recent induction with retinoic acid, were relatively resistant to effectors from all three mammalian species tested. Attempts to elicit a murine T-cell response to embryonal carcinoma by secondary in vitro stimulation failed. As previously shown, T killers directed to H-2 determinants were unable to lyse the MHC lacking embryonal carcinoma cells. The use of lectin "glues", a procedure supposedly capable of revealing the total cytotoxic. T-cell potential regardless of specificity, also failed to elicit lysis of these target cells. We have therefore failed to find evidence for T-cell recognition or lysis of embryonal carcinoma while NK cells from different species seemingly regard these latter cells as "universal" targets.

A murine teratocarcinoma stem cell line carries suppressed oncogenic virus genomes.

Murine teratocarcinoma stem cells are nonpermissive for productive infection by a variety of DNA (polyoma and SV40 virus) and RNA (murine leukemia and sarcoma virus) tumor viruses whereas differentiated murine cells derived from the stem cells are permissive for productive (or abortive in the case of SV40) infection by these same viruses. The block to productive infection by these oncogenic viruses is at a postpenetration step in the replication cycle of these viruses but the precise level of the block has not been established for any of these viruses. In this report we describe teratocarcinoma-derived stem and differentiated cell lines which should be especially useful in determining the level of the block to replication of ecotropic murine leukemia virus in murine teratocarcinoma stem cells. The stem cell line, OTT6050AF1 BrdU, which is completely nonpermissive to productive infection by Moloney murine leukemia virus and consists of 97% pluripotent stem cells, contains DNA copies of an RNA tumor virus which is indistinguishable from the N-tropic murine leukemia virus of AKR mice. The stem cells are negative for expression of viral reverse transcriptase, p30 and gp69/71 and no virus is found by XC plaque assay or other biological tests. Differentiated cells established from the same teratocarcinoma tumor are 100% positive for viral gp69/71, p30, and produce large amounts of reverse transcriptase activity and N-tropic virus as detected by biological assay. The virus isolated from the differentiated cells is closely related, if not identical to AKR N-tropic virus by nucleic acid hybridization studies and is thus not an endogenous virus of the 129 strain of mice. The teratocarcinoma tumor from which the cell lines were established had been carried in 129 mice and perhaps at some time in the mouse passage history the tumors were infected (nonproductively) with the N-tropic virus. Regardless of the origin of this viral DNA, the OTT6050A derived stem and differentiated cell lines should be extremely useful in defining in stem cells the step at which ecotropic murine leukemia virus replication is blocked.

Evidence for non-spliced SV40 RNA in undifferentiated murine teratocarcinoma stem cells

UNDIFFERENTIATED murine teratocarcinoma stem cells do not support simian vacuolating virus 40 (SV40) or polyoma virus (Py) replication or even the expression of the early papovavirus proteins. These undifferentiated cells are also entirely refractory to infection with ecotropic murine C-type viruses. If the stem cells are allowed to differentiate to a variety of somatic cell types, they become susceptible to infection by SV40, Py and ecotopic murine C-type viruses1–4. In contrast, adenovirus type 2 can infect and replicate in the undifferentiated stem cells, although not as efficiently as it can in the differentiated mouse cells5 and certainly much less efficiently than it does in human cells. Studies on the mechanism of the resistance of the undifferentiated teratocarcinoma cells to infection by SV40 has shown that the block is not at the level of virus adsorption, penetration uncoating or transport to the nucleus6. We present here evidence indicating that the absence of expression of SV40 genetic information in nondifferentiated teratocarcinoma cells reflects an inability to generate stable viral mRNA. More specifically, the refractoriness of these cells to SV40 seems to be correlated, at least in part, with a deficiency in the splicing of viral mRNA.

Squamous Cell Differentiation in a Clonal Teratocarcinoma Cell Line

Several clonal teratocarcinoma cell lines restricted to squamous cell differentiation have been established from a subculture of totipotential murine teratocarcinoma stem cells. These lines contain a continuum of cell types from less differentiated precursor cells to differentiated squamous cells. In contrast to their highly malignant progenitors, these cells are nontumorigenic. Chromosomally, the cells are near-tetraploid, and their DNA distributions are tetraploid. These cells lines may prove useful in the study of normal squamous cell differentiation and also will be important in studies concerning the conversion of malignant cells to non-malignant progeny.