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DNA methylation is not involved in the structural alterations ofOrnithine Decarboxylase or Total Chromatin of c-Ha-rasVal 12 Oncogene-Transformed NIH-3T3 Fibroblasts
Abstract
The ornithine decarboxylase (odc) gene is an early response gene, whose increased expression and relaxed chromatin structure is closely coupled to neoplastic growth. In various tumour cells, the odc gene displays hypomethylation at the sequences CCGG. Hypomethylation of genes is believed to correlate with chromatin decondensation and gene expression. Since a given pattern of DNA methylation may not be preserved in neoplastic cells, we studied the methylation status of odc gene at the CCGG sequences in c-Ha-rasVal 12 oncogene-transformed NIH-3T3 fibroblasts during the growth cycle and relative to their normal counterparts. We found that the methylation state of the odc gene and its promoter and mid-coding and 3' regions remain unaltered during the cell cycle. We also found that in ras oncogene-transformed cells, which display a more decondensed nucleosomal organization of chromatin than the normal cells, the CCGG sequences in bulk DNA and at the odc gene were methylated to the same extent as in the nontransformed cells. These data suggest that DNA hypomethylation at the CCGG sequences is not a prerequisite for chromatin decondensation and cell transformation by the c-Ha-rasVal 12 oncogene.
... As a conclusion, it is suggested that methylated CpG DNA sequences are not associated with the chromatin condensation enhancement that accompanies ras transformation in NIH 3T3 cells, since the enhanced amount of methylated DNA detected in the "condensed" chromatin of these cells was found to vary with the cell line considered and not to accompany the invariable amount of chromatin condensation of the transformed cell lines. This idea is supported by a report demonstrating that DNA methylation is not involved with the structural organization at the molecular level of the ornithine decarboxylase gene and the bulk chromatin in c-Ha-ras oncogene-transformed NIH 3T3 cells [14]. ...
Increased amounts of chromatin condensation (i.e., localized areas of high DNA density, or chromatin higher order packing state) have been described in NIH 3T3 cells transformed with the Ha-ras oncogene. The structural basis for this oncogene-mediated alteration in nuclear organization is unknown. Since DNA methylation is likely to be involved in regulating the nucleosomal level of DNA packaging, we studied the role of DNA methylation in higher-order chromatin organization induced by Ha-ras. CpG-methylated DNA content was estimated in "condensed" chromatin of Ha-ras-transformed NIH 3T3 cell lines which differ in ras expression and ras-induced metastatic ability but present approximately the same values of "condensed" chromatin areas. The question posed was that if DNA methylation were involved with the chromatin higher-order organization induced by Ha-ras in these cell lines, the methylated DNA density in the "condensed" chromatin would also be the same. The DNA evaluation was performed by video image analysis in Feulgen-stained cells previously subjected to treatment with Msp I and Hpa II restriction enzymes, which distinguish between methylated and non-methylated DNA. The amount of methylated CpG sequences not digested by Hpa II in "condensed" chromatin regions was found to vary in the studied ras-transformed cell lines. DNA CpG methylation status is thus suggested not to be involved with the higher order chromatin condensation induced by ras transformation in the mentioned NIH 3T3 cell lines.
... Micrococcal nuclease digestion of chromatin from parental and ras-transformed cells determined that the bulk chromatin, and chromatin at the immediate-early genes ornithine decarboxylase and c-myc was less condensed in the ras-transformed cells [16]. Experiments to determine the methylation status of DNA at the ornithine decarboxylase gene showed similar methylation levels in parental and transformed cells, thus it is not believed that hypomethylation plays a role in the formation of a relaxed chromatin structure in this case [88]. ...
Nuclear morphometric descriptors such as nuclear size, shape, DNA content and chromatin organization are used by pathologists as diagnostic markers for cancer. Tumorigenesis involves a series of poorly understood morphological changes that lead to the development of hyperplasia, dysplasia, in situ carcinoma, invasive carcinoma, and in many instances finally metastatic carcinoma. Nuclei from different stages of disease progression exhibit changes in shape and the reorganization of chromatin, which appears to correlate with malignancy. Multistep tumorigenesis is a process that results from alterations in the function of DNA. These alterations result from stable genetic changes, including those of tumor suppressor genes, oncogenes and DNA stability genes, and potentially reversible epigenetic changes, which are modifications in gene function without a change in the DNA sequence. DNA methylation and histone modifications are two epigenetic mechanisms that are altered in cancer cells. The impact of genetic (e.g., mutations in Rb and ras family) and epigenetic alterations with a focus on histone modifications on chromatin structure and function in cancer cells are reviewed here.
An altered nuclear morphology has been previously noted in association with Ras activation, but little is known about the structural basis, functional significance, signaling pathway, or reproducibility of any such change. We first tested the reproducibility of Ras-associated nuclear change in a series of rodent fibroblast cell lines. After independently developing criteria for recognizing Ras-associated nuclear change in a Papanicolaou stained test cell line with an inducible H(T24)-Ras oncogene, two cytopathologists blindly and independently assessed 17 other cell lines. If the cell lines showed Ras-associated nuclear change, a rank order of increasing nuclear change was independently scored. Ras-associated nuclear changes were identified in v-Fes, v-Src, v-Mos, v-Raf, and five of five H(T24)-Ras transfectants consisting of a change from a flattened, occasionally undulating nuclear shape to a more rigid spherical shape and a change from a finely textured to a coarse heterochromatic appearance. Absent or minimal changes were scored in six control cell lines. The two cytopathologists' independent morphologic rank orders were similar (P < .0002). The mitogen signaling pathway per se does not appear to transduce the change since no morphologic alterations were identified in cell lines with activations of downstream components of this pathway--MAPKK or c-Myc--and the rank orders did not correlate with markers of mitotic rate (P > .11). The rank order correlated closely with metastatic potential (P < .0014 and P < .0003) but not with histone H1 composition or global nuclease sensitivity. Based on published studies of five of the cell lines, there may be a correlation between increases in certain nuclear matrix proteins and the Ras-associated nuclear change.
Stimulation of the Ras-mitogen-activated protein kinase (MAPK) signal transduction pathway results in a multitude of events including expression of the immediate-early genes, c-fos and c-myc. Downstream targets of this stimulated pathway are the mitogen- and stress-activated protein kinases (MSK) 1 and 2, which are histone H3 kinases. In chromatin immunoprecipitation assays, it has been shown that the mitogen-induced phosphorylated H3 is associated with the immediate-early genes and that MSK1/2 activity and H3 phosphorylation have roles in chromatin remodeling and transcription of these genes. In oncogene-transformed fibroblasts in which the Ras-MAPK pathway is constitutively active, histone H1 and H3 phosphorylation is increased and the chromatin of these cells has a more relaxed structure than the parental cells. In this review we explore the deregulation of the Ras-MAPK pathway in cancer, with an emphasis on breast cancer. We discuss the features of MSK1 and 2 and the impact of a constitutively activated Ras-MAPK pathway on chromatin remodeling and gene expression.
To evaluate the function of the murine ornithine decarboxylase (ODC) gene promoter, expression of chimeric ODC-chloramphenicol acetyltransferase (CAT) plasmids (pODCcat) containing 1,658 nt of the ODC promoter sequence and its various 5'-deletions was analyzed. In transient expression assays with NIH/3T3 mouse cells, pODCcat constructs exhibited fairly strong promoter activity yielding CAT values up to 40% of those obtained with the viral promoter RSV. Interestingly, 5'-deletions of the pODCcat constructs increased the promoter activity over that achieved using the entire 1.6-kb 5'-flanking region, with the highest activity being observed with about 750 nt of the ODC promoter. This finding suggests that the distal part of the promoter includes DNA elements which are involved in repressing its function. The promoter region could be deleted down to the proximal 97 nt and still be stimulated by cAMP to the same extent as the 1.6-kb promoter. DNase I footprinting and methylation interference studies showed that a specific protein binds to the region from -59 to -39, which encompasses a DNA motif resembling the consensus cyclic AMP response element (CRE). However, comparative gel retardation and Southwestern blotting experiments with the putative ODC-CRE and the somatostatin promoter CRE indicated that the 70-kDa protein interacting with the CRE-like element of the ODC promoter is different from the well-characterized nuclear CRE-binding protein CREB.
The ornithine decarboxylase (ODC)-deficient Chinese hamster ovary (CHO) cell line C55.7 has normal amounts of ODC mRNA with very low amounts of immunologically detectable ODC protein, suggesting a structural mutation; however, 5-azacytidine treatment leads to phenotypical reversion (Steglich, C., and Scheffler, I. E. (1985) Somat. Cell Mol. Genet. 11, 11-23). We have demonstrated by chemical cleavage a single base mismatch in DNA heteroduplexes composed of wild-type and mutant cDNA strands. DNA sequencing showed that the mutant phenotype results from an aspartate-glycine substitution at amino acid 381 of the protein. When 5-azacytidine-revertant cell lines were selected for resistance to alpha-difluoromethylornithine, the resulting amplified ODC gene was structurally indistinguishable from the wild type gene. These results suggested the existence of a single active ODC locus in CHO cells. Using the methylation-sensitive restriction endonucleases AvaI and HpaII, we found evidence for two differentially methylated alleles in wild type, ODC-deficient and alpha-difluoromethylornithine-resistant cells. One of the alleles appeared completely inactivated by hypermethylation but could be reactivated by demethylation in spontaneous or 5-azacytidine-induced revertants.
We have compared the nucleosomal organization of c-Ha-rasVal 12 oncogene-transformed NIH-3T3 fibroblasts with that of normal fibroblasts by using micrococcal nuclease (MNase) as a probe for the chromatin structure. The bulk chromatin from asynchronously and exponentially growing ras-transformed cells was much more sensitive to MNase digestion than chromatin from the normal cells. Southern hybridization analyses of the MNase digests with probes specific for the ornithine decarboxylase (odc) and c-myc genes showed that the coding and/or 3' end regions of these growth-inducible genes carry a nucleosomal organization both in ras-transformed and normal cells. Studies with cells synchronized by serum starvation showed that in both cell lines the nucleosomal organization of chromatin is relatively condensed at the quiescent state, becomes highly decondensed during the late G1 phase of the cell cycle, and starts again to condense during the S phase. However, in ras-transformed cells the decondensation state stayed much longer than in normal cells. Moreover, irrespective of the phase of the cell cycle the bulk chromatin as well as that of the odc and c-myc genes was more sensitive to MNase digestion in the ras-transformed cell than in the normal fibroblast. Decondensation of the chromatin was also observed in the normal c-Ha-ras protooncogene-transfected cells, but to a lesser extent than in the mutant ras-transformed cells. Whether the increased degree of chromatin decondensation plays a regulatory role in the increased expression of many growth-related genes in the ras-transformed cells remains an interesting object of further study.
We have studied the effects of c-Ha-ras oncogene in mouse NIH 3T3 fibroblasts by DNA transfection and analysis of gene expression at the mRNA and protein level in a heat- and heavy metal-inducible model system. The human c-Ha-ras proto-oncogene and oncogene were cloned under the hsp70 heat-shock promoter. Clonal lines of cells with negligible basal expression of the hsp-c-Ha-ras oncogene construct were chosen on the basis of the inducibility of p21c-Ha-ras protein and several transformation parameters. We demonstrate that the expression of ornithine decarboxylase (ODC) mRNA is enhanced approximately 4-6 h after the induction of the p21c-Ha-ras oncoprotein. This increase was reversible upon cessation of c-Ha-ras mRNA and protein synthesis, while constitutively elevated ODC was characteristic for stably c-Ha-ras-transformed cells. The high-level expression of ODC in ras-transformed cells was insensitive to tumour promoter stimulation. A similar mRNA induction by c-Ha-rasVal-12 was also observed for two other serum- and tumour promoter-regulated genes associated with the transformed phenotype: transin (stromelysin) and the glucose transporter. This prompted us to examine also potential changes in the expression of the serum- and tumour promoter-induced transcription factor genes junB and c-jun after induction of the hsp--c-Ha-ras construct. The junB mRNA was enhanced approximately 10-fold and the c-jun oncogene mRNA to a lesser degree in the hsp--c-Ha-ras-transfected cells after zinc activation of the hsp70 promoter. These effects were not seen in similarly treated control cells.(ABSTRACT TRUNCATED AT 250 WORDS)
We have studied the ability of the neu tyrosine kinase to induce a signal for the activation of cell growth-regulated genes. Serum-starved NIH 3T3 cells expressing an epidermal growth factor receptor (EGF-R)/neu construct encoding a hybrid receptor protein were stimulated with EGF and the activation of the neu tyrosine kinase and stimulation of growth factor inducible genes were followed at the mRNA, protein, and activity levels, and compared to the corresponding responses in the neu proto-oncogene and oncogene expressing cells. Induction of the expression of jun mRNAs was an immediate early effect of EGF stimulation, followed by a marked increase in the biosynthesis of the fos/jun transcription factor complex and an increased transcription factor activity as measured by a recombinant transcription unit using chloramphenicol acetyltransferase assays. In distinction, elevated AP-1/PEA-1 activity in the absence of a significant increase in jun and fos expression was characteristic of the neu oncogene-expressing cells. The glucose transporter mRNA increased at 2 h of EGF stimulation and was associated with enhanced glucose transport of the EGF-treated cells. An increase of ornithine decarboxylase (ODC) mRNA and activity followed these changes. In contrast, serum-starved, EGF-treated neu proto-oncogene- and oncogene-expressing cells showed constitutively low and high glucose transporter and ODC activities, respectively. These findings demonstrate that the chimeric EGF-R/neu receptor is capable of activating the expression of both immediate early genes and biochemical activities associated with cell growth stimulation.
We have isolated and sequenced a full-length cDNA clone encoding rat glyceraldehyde-3-phosphate-dehydrogenase (GAPDH, E.C.1.2.1.12).
The entire mRNA is 1269 nucleotides long exclusive of poly(A) and contains respectively 71 and 196 bases of 5′ and 3′ non-coding
regions. Primer extension as well as S1 nuclease protection experiments clearly established that a single (or at least a highly prominent) GAPDH mRNA species is
expressed in all rat tissues examined. This sequence allowed the determination of the hitherto unknown primary structure of
rat GAPDH which is 333 aninoacids long. Comparison between GAPDH sequences from rat, man and chicken revealed a high degree
of sequence conservation at both nucleotide and protein levels.
Methylated DNA-binding protein (MDBP) from human placement recognizes specific DNA sequences containing 5-methylcytosine (m5c) residues. Comparisous of binding of various prokaryotic DNAs to MDBP indicate that m5CpG is present in the recognition sites for this protein but is only part of the recognition sequence. Specific binding to
MDBP was observed for bacteriophage XP12 DNA, which naturally contains ∼/3 of its residues as m5C, and for Micrococcus luteus DNA, M13mp8 replicative form (RF) DNA, and pBR322 when these three DNAs were methylated at CpG sites by human DNA methyltransferense.
Five DNA regions binding to MDBP have been localized by DNase I footprinting or restriction mapping in methylated pBR322 and
M13mp8 RF DNAs. A comparison of their sequences reveals a common 5'-m5CGRm5CG-3' element or closely related sequence in which one of the m5residues may be replaced by a T. In addition to this motif, one upstream and one downstream m5CpG as well as other common residues over an ∼20-bp long region may be recognized by MDBP.
With the use of the isoschizomeric restriction endonucleases HpaII and MspI, we found that mouse tumour ornithine decarboxylase (ODC; EC 4.1.1.17) genes are extensively methylated. ODC genes in L1210 mouse leukaemia cells were apparently more methylated than in Ehrlich ascites carcinoma, as revealed by the use of HpaII endonuclease, yet the digestion of genomic DNA isolated from these two murine tumour cell lines with MspI, which cleaves at a CCGG sequence, also with internally methylated cytosine, resulted in an apparently identical restriction pattern. It is possible that the amplification of ODC genes in Ehrlich ascites-carcinoma cells in response to 2-difluoromethylornithine (DFMO) was associated with hypomethylation, or that less-methylated genes were amplified. A human myeloma (Sultan) cell line only revealed three separate hybridization signals when cleaved with HpaII. One of these signals was amplified under the pressure of DFMO. When cleaved with MspI, these three HpaII fragments disappeared and were replaced by a double signal of 2.3-2.4 kilobase-pairs (kbp) in size. The amplified ODC sequences in the Sultan myeloma cell line apparently originated from chromosome 2, as indicated by a unique hybridization signal in a 5.8 kbp HindIII fragment specific for the human ODC locus on chromosome 2. A comparison of different human cells, the Sultan myeloma, a lymphocytic B-cell leukaemia (Ball), normal mononuclear leucocytes and leucocytes obtained from leukaemia patients, revealed interesting differences in the methylation of ODC genes. The use of two restriction endonucleases (HpaII and CfoI), the cleavage site for both of which contains a CG sequence and which only cleave when cytosine is unmethylated, indicated that ODC genes in the lymphocytic leukaemia cells were much less methylated than those in the normal leucocytes or in the Sultan cells.
NIH 3T3 cells transformed with the human c-Ha-rasVal-12 oncogene showed markedly enhanced activity of ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis, as compared with their nontransformed counterparts. While in normal and in c-Ha-ras proto-oncogene-transfected cells stimulation with serum caused a transient induction of ODC, in cells transfected with the mutant c-Ha-ras oncogene the activity of ODC persisted at high levels for greatly extended periods of time. The amounts of immunoreactive ODC protein roughly paralleled the changes in the enzyme activity. The augmentation of ODC content by transformation could be largely, but not solely, accounted for by an enhanced accumulation of ODC mRNA. Nuclear run-off transcription assays demonstrated that in transformed cells the rate of transcription of the ODC gene was increased but to a much lower extent than the increase in the level of ODC mRNA. The turnover of ODC mRNA, as measured after actinomycin D treatment, was negligible in transformed cells for up to 8 h, whereas in normal cells the messenger content was initially decreased, by 40% within 4 h, and then remained constant. In normal cells, however, actinomycin D depressed the expression of ODC by more than 80%, while in transformed cells the activity of ODC was slightly superinduced, corresponding to the changes of ODC mRNA. These findings suggest that labile proteins may be involved in the regulation of both the stability and translatability of the ODC mRNA. Transformation led also to about 3-fold stabilization of ODC as determined by an exposure of the cells to cycloheximide. The results thus suggest ODC deregulation at multiple levels in the ras-oncogene-transformed cells.
Mouse ornithine decarboxylase (ODC) genomic clones were isolated from a bacteriophage lambda genomic library representing mouse myeloma 653-1 cells which over-produce ODC due to amplification of an active ODC gene. Sequence analysis of the amplified ODC gene revealed that ODC mRNA is encoded by 12 exons, 10 of which (exons 3 to 12) code for the ODC protein. Exon 12 also corresponds to the 3' noncoding region of the two species of ODC mRNA which are formed by alternative utilization of two polyadenylation signals separated from each other by 422 nucleotides. The transcription initiation site was mapped by S1 nuclease protection and by primer extension analysis. The 5' flanking region is extremely rich in G + C and contains typical promoter motifs such as the TATA box and SP1 transcription factor binding sites. Joining the 5' flanking region to the Escherichia coli chloramphenicol acetyltransferase structural gene and its introduction into mouse cells resulted in the expression of a high level of chloramphenicol acetyltransferase activity. Comparing the sequence of the ODC gene to our previously published sequence of ODC cDNA revealed a disagreement between the sequences located 5' to the AvaI site and demonstrated that this region of our previously reported cDNA represents a cloning artifact. The portion of the correct 5' noncoding region encoded by exon 1 is extremely rich in G + C and includes potential secondary structures which may be involved in translational regulation of ODC mRNA.
Ornithine decarboxylase (OrnDCase; L-ornithine carboxy-lyase, EC 4.1.1.17) mRNA present in mouse kidney comprises two species with molecular sizes of approximately 2.2 and approximately 2.7 kilobases (kb). cDNA clones prepared from murine kidney OrnDCase mRNA were used to determine the reason for the size heterogeneity of these mRNAs. Two of the cDNA clones (pODC16 and pODC74) that differed at the 3' termini were isolated and sequenced. DNA sequencing indicated that each cDNA had a poly(A) tail; however, pODC74 was 429 nucleotides longer than pODC16 at the 3' end and contained two AATAAA signals for poly(A) addition. That the longer cDNA corresponded to the larger mRNA was confirmed by hybridization of a unique Pst I/Pst I fragment from the 3' terminus of pODC74 only to the 2.7-kb OrnDCase mRNA. The two cDNAs did not represent full-length copies of OrnDCase mRNAs and were 1199 (pODC16) and 1204 base pairs (bp) (pODC74) long. There were five mismatches in their 759-bp-long overlapping nucleotide sequence, suggesting that the 2.2- and 2.7-kb OrnDCase mRNAs may be products of two separate, yet very similar, OrnDCase genes. Androgen regulation of the accumulation of these two OrnDCase mRNAs appeared to occur coordinately, as testosterone administration brought about comparable increases in their concentrations in mouse kidney.
In an earlier study it was discovered that when Friend erythroleukemia cells (FELC) were exposed to a variety of chemical agents capable of inducing differentiation, their DNA underwent genome-wide transient demethylation. In an attempt to elucidate the biochemical mechanism responsible for this phenomenon we have induced FELC with 5 mM hexamethylenebisacetamide and labeled the DNA in vivo with a density label, 5-bromodeoxyuridine, and a radioactive label, deoxy[5-3H]cytidine. Newly replicated DNA (heavy-light) was separated from parental DNA (light-light) by isopycnic centrifugation. Incorporation of deoxy[5-3H]cytidine into light-light duplex DNA has been observed only in induced cells concomitantly with the demethylation of the DNA, whereas, in parallel experiments, deoxy[G-3H]adenosine was not incorporated into light-light DNA. It was also found that the labeling of light-light DNA with deoxy[5-3H]cytidine is transient since the 3H label was removed from the DNA during the period of de novo DNA methylation that follows the demethylation. These results, taken together, strongly suggest that the demethylation of the DNA during differentiation is achieved by an enzymatic mechanism whereby 5-methylcytosine is replaced by cytosine.
DNA methylase activity in the nuclei of somatic cells arrested at G0 increased markedly when the cells were subjected to a mitogenic stimulus. Treatment of mouse splenocytes with Concanavalin A resulted in about 20-fold increase in methylase activity within 20 h starting 12-15 h after Concanavalin A addition. The methylase level in rat liver was elevated approximately 3-fold at about 20-h posthepatectomy. A detailed time course of the increase in methylase activity with respect to the cell cycle revealed that the onset of this event coincided with the entry of the cells into S phase. In both systems, the extent of methylation in CpG sequences is not altered significantly even under conditions of active DNA synthesis which is induced by the mitogenic effect. These results suggest that the cell responds to the mitogenic stimulus by adjusting the DNA methylase activity to enable conservation of the methylation level in DNA.
5-Azacytidine (azaC), a drug that induces decreased methylation of DNA in mammalian cells, was shown previously to induce differentiation of mesenchymal cell types in CHEF/18 cells (Chinese hamster embryo fibroblasts). This paper describes the effectiveness of azaC in inducing tumorigenicity in CHEF/18 cells, previously shown to be nontumorigenic stable diploids. A short exposure of growing cells to 3 microM azaC induced tumor-forming ability in CHEF/18 stem cells. Pre-adipocyte clones and subclones derived from CHEF/18 by prior treatment with azaC were also found to be tumorigenic. Pre-adipocytes previously induced by insulin in the absence of azaC were mostly nontumorigenic, but one clone produced tumors and gave rise to both tumorigenic and nontumorigenic subclones. Karyotype analysis of 41 clones and subclones from azaC-induced and insulin-induced pre-adipocytes revealed a complete correlation between tumor-forming ability and the presence of trisomy for chromosome 3q. In addition, the tumorigenic and tumor-derived lines were demethylated at specific C-C-G-G sites in the preproinsulin, Ha-ras, and Ki-ras genes as revealed by blot hybridization to Msp I- and Hpa II-digested DNAs, whereas the nontumorigenic lines resembled the CHEF/18 controls. This three-way correlation between tumorigenicity, trisomy for 3q, and specific demethylation suggests that decreased DNA methylation may be involved both in differentiation and in tumorigenicity, and that azaC may induce chromosomal aberrations as well as altering DNA methylation.
F9 teratocarcinoma cells can be grown as monolayers or aggregates, and upon treatment with retinoic acid they will differentiate into parietal or visceral endoderm, respectively. Visceral endoderm specifically synthesizes alpha-fetoprotein and albumin mRNAs, which are not found in parietal endoderm. In contrast, both endoderms produce enhanced levels of the major histocompatibility antigen (H2) mRNA compared with F9 cells. F9 cells contain highly methylated DNA as judged by restriction enzyme digestion. However, upon differentiation into visceral endoderm, there is a genome-wide loss of methylation in induced, silent, and constitutively expressed genes. Experiments in which methylation loss is induced via the methyltransferase inhibitor 5-azacytidine result in no induction of alpha-fetoprotein mRNA and no morphological differentiation, suggesting that methylation loss alone is not sufficient to induce the visceral endoderm phenotype. Likewise, 5-azacytidine treatment of differentiated cells does not result in enhanced expression of alpha-fetoprotein mRNA. However, the patterns of loss of DNA methylation at all sites examined after differentiation or 5-azacytidine treatment were remarkably similar, suggesting that the two occur by a similar mechanism, the inhibition of DNA methyltransferase activity. These results argue that the specificity for methylation loss at a given site is an inherent property of aggregated F9 cell chromatin. This system provides a model for studying a tissue-specific change in DNA methylation upon differentiation.
The overall 5-methylcytosine (m5C) content of DNA from normal tissues varies considerably in a tissue-specific manner. By high-performance liquid chromatography,
we have examined the (m5C) contents of enzymatic digests of DNA from 103 human tumors including benign, primary malignant and secondary malignant
neoplasms. The diversity and large number of these tumor samples allowed us to compare the range of DNA methylation levels
from neoplastic tissues to that of normal tissues from humans. Most of the metastatic neoplasms had significantly lower genomic
(m5C) contents than did most of the benign neoplasms or normal tissues. The percentage of primary malignancies with hypomethylated
DNA was intermediate between those of metastases and benign neoplasms. These findings might reflect an involvement of extensive
demethylation of DNA in tumor progression. Such demethylation could be a source of the continually generated cellular diversity
associated with cancer.
DNA-mediated gene transfer was used to investigate the mode of inheritance of 5-methylcytosine in mouse L cells. Unmethylated phi X174 replicative form DNA remains unmethylated after its introduction and integration into these cells. On the other hand, phi X174 replicative form DNA that was methylated in vitro at its C-C-G-G residues retains these methylations as shown by restriction enzyme analysis with Hpa II and Msp I to detect methylation at this specific site. Although these unselected methylated vectors are prone to lose 30-40% of their methyl moieties upon transfection, this demethylation appears to be random. Once established, the resulting methylation pattern is stable for at least 100 cell generations. In order to examine the specificity of methylation inheritance, fully hemimethylated duplex phi X174 DNA was synthesized in vitro from primed single-strand phi X174 DNA by using 5-methyl deoxycytidine 5'-triphosphate. This molecule was inserted into mouse L cells by cotransformation and subsequently was analyzed by a series of restriction enzymes. Only methylations located at C-G residues were conserved after many generations of cell growth. The results suggest that the inheritance of the cellular DNA methylation pattern is based on a C-G-specific methylase that operates on newly replicated hemimethylated DNA.
It is now an established fact that the distribution of methylated bases along DNA form a distinct pattern that is characteristic of the cell in which this DNA is harbored. It is also becoming clear that the biological functions that are fulfilled by DNA methylation are dependent on these patterns. The relationship between DNA methylation patterns and the various biological functions that are associated with these patterns is discussed in other chapters in this volume. However, it is appropriate to mention one familiar, clear-cut example of how a pattern of methylation exerts its biological function. The biological function of the restriction-modification systems in bacteria is to protect the cell from invasion of foreign genetic material. This protection is achieved via degradation of foreign DNA by restriction enzymes that are sequence-specific endonucleases. The DNA cleavage activity of these endonucleases is refractory to methylation at their recognition sites (see Chapter 5). The methylation is carried out by sequence-specific modification enzymes, which can methylate efficiently the restriction enzyme recognition sites of the host DNA. Since this methylation must provide protection to all sites in the host DNA, the pattern of methylation obtained in this case is determined by the distribution of the restriction sites along the DNA.
Ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis, becomes upregulated during cell proliferation and transformation. Here we show that intact ODC activity is needed for the acquisition of a transformed phenotype in rat 2R cells infected with a temperature-sensitive mutant of Rous sarcoma virus. Addition of the ODC inhibitor alpha-difluoromethyl ornithine (DFMO) to the cells (in polyamine-free medium) before shift to permissive temperature prevented the depolymerization of filamentous actin and morphological transformation. Polyamine supplementation restored the transforming potential of pp60v-src. DFMO did not interfere with the expression of pp60v-src or its in vitro tyrosine kinase activity. The tyrosine phosphorylation of most cellular proteins, including ras GAP, did not either display clear temperature- or DFMO-sensitive changes. A marked increase was, however, observed in the tyrosine phosphorylation of phosphatidylinositol 3-kinase and proteins of 33 and 36 kD upon the temperature shift, and these hyperphosphorylations were partially inhibited by DFMO. A DFMO-sensitive increase was also found in the total phosphorylation of calpactins I and II. The well-documented association of GAP with the phosphotyrosine-containing proteins p190 and p62 did not correlate with transformation, but a novel 42-kD tyrosine phosphorylated protein was complexed with GAP in a polyamine- and transformation-dependent manner. Further, tyrosine phosphorylated proteins of 130, 80/85, and 36 kD were found to coimmunoprecipitate with pp60v-src in a transformation-related manner. Altogether, this model offers a tool for sorting out the protein phosphorylations and associations critical for the transformed phenotype triggered by pp60v-src, and implicates a pivotal role for polyamines in cell transformation.
We have studied the ability of the neu tyrosine kinase to induce a signal for the activation of cell growth-regulated genes. Serum-starved NIH 3T3 cells expressing an epidermal growth factor receptor (EGF-R)/neu construct encoding a hybrid receptor protein were stimulated with EGF and the activation of the neu tyrosine kinase and stimulation of growth factor inducible genes were followed at the mRNA, protein, and activity levels, and compared to the corresponding responses in the neu proto-oncogene and oncogene expressing cells. Induction of the expression of jun mRNAs was an immediate early effect of EGF stimulation, followed by a marked increase in the biosynthesis of the fos/jun transcription factor complex and an increased transcription factor activity as measured by a recombinant transcription unit using chloramphenicol acetyltransferase assays. In distinction, elevated AP-1/PEA-1 activity in the absence of a significant increase in jun and fos expression was characteristic of the neu oncogene-expressing cells. The glucose transporter mRNA increased at 2 h of EGF stimulation and was associated with enhanced glucose transport of the EGF-treated cells. An increase of ornithine decarboxylase (ODC) mRNA and activity followed these changes. In contrast, serum-starved, EGF-treated neu proto-oncogene- and oncogene-expressing cells showed constitutively low and high glucose transporter and ODC activities, respectively. These findings demonstrate that the chimeric EGF-R/neu receptor is capable of activating the expression of both immediate early genes and biochemical activities associated with cell growth stimulation.
Transient transfection into L8 myoblasts has been used to study the rat alpha-actin gene promoter. Demodification of specific sites occurs in two stages, with a hemimethylated intermediate formed within a few hours after entry of the alpha-actin gene construct into the cell. The removal of the methyl moiety from the complementary strand takes place after a delay of at least 48 hr, and both events are actively carried out in the absence of DNA replication. By assaying gene activity during the course of the transfection, it was possible to demonstrate that demethylation of both strands at the critical CpG loci is essential to activate transcription. Genetic analysis revealed the existence of cis-acting elements required for demethylation. The recognition of these sites early in the differentiation process probably leads to the demodification events required to make the gene accessible to its transcription factors.
The 5-methylcytosine residues of L-cells have been labeled with [methyl-3H]-L-methionine and their chromatin localization studied using deoxyribonucleases. The kinetics of micrococcal nuclease digestion showed that the methylated cytosine residues are concentrated within regions resistant to nuclease digestion and preferentially missing from those regions between nucleosomes which are nuclease sensitive. Using DNA hybridization kinetic analysis, it is shown that 5-methylcytosine is abundant in highly repeated sequences but is also present in middle repetitive and unique sequence DNA.
The enzyme ornithine decarboxylase is the key regulator of the synthesis of polyamines which are essential for cell proliferation. Expression of this enzyme is transiently increased upon stimulation by growth factors, but becomes constitutively activated during cell transformation induced by carcinogens, viruses or oncogenes. To test whether ornithine decarboxylase could be a common mediator of transformation and oncogenic itself, we transfected NIH3T3 cells with expression vectors carrying the complementary DNA encoding human ornithine decarboxylase in sense and antisense orientations. The increased expression of the enzyme (50-100-times endogenous levels) induced not only cell transformation, but also anchorage-independent growth in soft agar and increased tyrosine phosphorylation of a protein of M(r) 130K. Expression of ornithine decarboxylase antisense RNA was associated with an epithelioid morphology and reduced cell proliferation. Moreover, blocking the endogenous enzyme using specific inhibitor or synthesizing antisense RNA prevented transformation of rat fibroblasts by temperature-sensitive v-src oncogene. Our results imply that the gene encoding ornithine decarboxylase is a proto-oncogene central for regulation of cell growth and transformation.
Direct genomic sequencing revealed that cytosine residues known to have undergone a germ-line mutation in the low density lipoprotein receptor gene or somatic mutations in the p53 tumor suppressor gene were methylated in all normal human tissues analyzed. Thus, these mutations should be scored as transitions from 5-methylcytosine to thymine rather than from cytosine to thymine. Methylated cytosines occur exclusively at CpG dinucleotides, which, although markedly underrepresented in human DNA, are sites for more than 30 percent of all known disease-related point mutations. Thus, 5-methylcytosine functions as an endogenous mutagen and carcinogen in humans, in that methylation seems to increase the potential for mutation at cytosine residues at least by a factor of 10.
The ornithine decarboxylase (ODC; EC 4.1.1.17) gene in parental, dexamethasone-resistant and 2-difluoromethylornithine (DFMO)-resistant human IgG-myeloma-cell lines was studied with the aid of methylation-sensitive restriction endonucleases and probes recognizing different parts of the gene. In all cell lines the promoter region of the ODC gene appeared to be heavily methylated, whereas the first long intron was unmethylated. Methylation analyses of several clones from the parental cell line revealed that these cells are heterogeneous with respect to the methylation status of the ODC gene, whereas all clones from DFMO-resistant cell lines displayed the same methylation pattern. Two of the parental clones represented a hypomethylated type very close to that exclusively found among the DFMO-resistant clones with ODC gene amplification. This typical methylation pattern was due to decreased methylation of a few CCGG sequences in the 3'-flanking region of the gene. It is possible that this kind of hypomethylation favours the initiation of the gene-amplification process in certain individual cells. This hypothesis was supported by the finding that no hypomethylation was present in the ODC gene of another human myeloma cell line that had acquired resistance to DFMO without gene amplification. In a dexamethasone-resistant cell line that overproduced ODC mRNA at normal gene dosage there were some minor differences between the methylation pattern of the ODC gene of different clones, but no such hypomethylation could be found in clones from the parental cell line. In dexamethasone-resistant cells the ODC gene was hypomethylated around the two HpaII sites and three CfoI sites in the coding region and also, as well as in cells with amplified ODC sequences, in the 3'-flanking region of the gene. Some hypomethylation in the distant 5'-flanking region was also observed.
The stimulation of quiescent murine fibroblasts by growth factors and by phorbol esters results in a rapid and transient transcriptional activation of a large group of so-called immediate early genes. Several such genes were found to be induced in chicken embryo fibroblasts following activation of a temperature sensitive (ts) Rous sarcoma virus v-src mutant following temperature shift (Simmons et al., 1989). In contrast, the classical immediate early genes c-myc, c-fos and c-jun were essentially uninducible upon activation of a ts v-src mutant in rat-1 fibroblasts (Welham et al., 1990). We have cloned 9 cDNAs of genes that are rapidly and transiently inducible in rat fibroblasts by ts v-src mutants, and by a ts Fujinami sarcoma virus v-fps mutant. Six of these cDNAs are derived from the known immediate early genes NGFI-A, KC, c-fos, tissue factor, PC4 and ornithine decarboxylase; the other three cDNAs have not been described before. These 9 genes showed individual profiles of inducibility by fetal calf serum, epidermal growth factor (EGF) and by phorbol esters. Their response to the retroviral oncogenic protein-tyrosine kinases correlated best with the one to EGF, suggesting a common pathway of signal transduction. c-fos did not respond strongly to this pathway but was well induced by fetal calf serum. NGFI-A, however, was induced to a similar extent by all activators tested. Furthermore, we demonstrated that the induction of several of these genes by the retroviral oncogenic protein-tyrosine kinases is rapid, direct and occurs at the transcriptional level.
CpG islands are normally methylation free in cells of the animal, even when the associated gene is transcriptionally silent. In mouse NIH 3T3 and L cells, however, over half of the islands are heavily methylated. Near identity of the methylated subset in the two cell lines suggested that methylation is confined to genes that are nonessential in culture. In agreement with this, islands at several tissue-specific genes, but not at housekeeping genes, have become methylated in many human and mouse cell lines. At the chromatin level, methylated islands are Mspl resistant compared with their nonmethylated counterparts. We suggest that mutation-like gene inactivation due to CpG island methylation is widespread in many cell lines and could explain the loss of cell type-specific functions in culture.
An understanding of the function and control of DNA methylation in eukaryotes has been elusive. Studies of Neurospora crassa have led to a model that accounts for the chromosomal distribution of methylation and suggests a basic function for DNA methylation in eukaryotes.
Initiation of tumorigenesis in mouse skin can be accomplished by mutagenesis of the H-ras gene by treatment with chemical carcinogens. A mouse model system has been developed to study the additional genetic events that take place during tumor progression. Skin carcinomas were induced in F1 hybrid mice exhibiting restriction fragment length polymorphisms at multiple chromosomal loci. Analysis of loss of heterozygosity in such tumors showed that imbalance of alleles on mouse chromosome 7, on which the H-ras gene is located, occurs very frequently in skin carcinomas. The chromosomal alterations detected, which included both nondisjunction and mitotic recombination events, were only seen in tumors that have activated ras genes. We conclude that gross chromosomal alterations that elevate the copy number of mutant H-ras and/or lead to loss of normal H-ras are a consistent feature of mouse skin tumor development.
Actively transcribed chromatin is structurally different from bulk inactive chromatin. It has been difficult to define the molecular basis of the difference, however, because purified fractions of active chromatin were not available. We have overcome this problem by releasing oligonucleosomes from the nonmethylated CpG-rich islands (CpG islands) of HeLa cell nuclei using restriction endonucleases. Since CpG islands very often include the promoters and 5' transcribed regions of genes, they represent a model for the "active" chromatin structure. CpG island chromatin differs in three respects from bulk chromatin prepared in the same way: histone H1 is present in very low amounts; histones H3 and H4 are highly acetylated; and nucleosome-free regions are present. Except for the latter regions, the average nucleosomal spacing is similar to that of bulk chromatin.
We have compared nuclear accessibility of methylated and nonmethylated sequences using restriction enzymes. MspI, which cuts CpG sites in naked DNA regardless of methylation, cut DNA in intact mouse liver or brain nuclei almost exclusively at CpG islands. Bulk chromatin was not significantly cleaved by MspI but was cleaved extensively by enzymes that do not recognize CpG. Quantitative analysis of limit digests showed that MspI and another methyl-CpG insensitive enzyme, Tth, have a strong bias against cutting methylated sites in these nuclei. Southern analysis confirmed this at three genomic loci. Our results suggest that resistance to nucleases is mediated by factors that are bound specifically to methylated CpGs. MeCP, a protein that binds to methylated DNA in vitro, may be one such factor, since nuclease resistance was significantly reduced in an MeCP-deficient cell line.
Previous investigations showed that inhibition of DNA synthesis by hydroxyurea, aphidicolin, or 5-fluorodeoxyuridine produced large changes in the composition and nucleosome repeat lengths of bulk chromatin. Here we report results of investigations to determine whether the changes in nucleosome repeat lengths might be localized in the initiated replicons, as postulated [D'Anna, J. A., & Prentice, D. A. (1983) Biochemistry 22, 5631-5640]. In most experiments, Chinese hamster (line CHO) cells were synchronized in G1, or they were synchronized in early S phase by allowing G1 cells to enter S phase in medium containing 1 mM hydroxyurea or 5 micrograms mL-1 aphidicolin, a procedure believed to produce an accumulation of initiated replicons that arise from normally early replicating DNA. Measurements of nucleosome repeat lengths of bulk chromatin, the early replicating unexpressed metallothionein II (MTII) gene region, and a later replicating repeated sequence indicate that the changes in repeat lengths occur preferentially in the early replicating MTII gene region as G1 cells enter and become synchronized in early S phase. During that time, the MTII gene region is not replicated nor is there any evidence for induction of MTII messenger RNA. Thus, the results are consistent with the hypothesis that changes in chromatin structure occur preferentially in the early replicating (presumably initiated) replicons at initiation or that changes in chromatin structure can precede replication during inhibition of DNA synthesis. The shortened repeat lengths that precede MTII replication are, potentially, reversible, because they become elongated when the synchronized early S-phase cells are released to resume cell cycle progression.
The methylation state of DNA from human colon tissue displaying neoplastic growth was determined by means of restriction endonuclease
analysis. When compared to DNA from adjacent normal tissue, DNA from both benign colon polyps and malignant carcinomas was
substantially hypomethylated. With the use of probes for growth hormone, gamma-globin, alpha-chorionic gonadotropin, and gamma-crystallin,
methylation changes were detected in all 23 neoplastic growths examined. Benign polyps were hypomethylated to a degree similar
to that in malignant tissue. These results indicate that hypomethylation is a consistent biochemical characteristic of human
colonic tumors and is an alteration in the DNA that precedes malignancy.
Association of alteration in DNA methylation pattern in triggering 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced transcription of ornithine decarboxylase (ODC) gene in T24 cells was determined. In accord with our previous findings (Archiv. Biochem. Biophys., 262: 326-336, 1988), TPA treatment of T24 cells, cultured in serum-free medium, resulted in a dramatic (approximately 15-fold) increase in ODC activity which was accompanied by a proportional increase in hybridizable amount of ODC mRNA. Data from nuclear run-off transcription assay revealed that TPA-induced accumulation of ODC mRNA is the result of increased transcription initiation. Since DNA hypomethylation has been proposed to be a mechanism involved in the regulation of transcription of some gene(s), we examined the changes in the methylation patterns in the ODC gene isolated from the vehicle (ethanol)- and TPA-treated T24 cells. The autoradiograms resulting from the Southern blot analysis of DNA cleaved with several methylation-sensitive restriction endonucleases [e.g., HpaII, MspI, cfoI (HhaI), SalI, XhoI] exhibited no difference in methylation pattern of ODC gene in T24 cells. Also, a single or chronic application of TPA to either noninitiated or 7,12-dimethylbenz(a)anthracene-initiated mouse skin failed to alter DNA methylation pattern of ODC gene. Furthermore, the hypomethylation agent 5-azacytidine failed to induce ODC mRNA in T24 cells. These results indicate that TPA does not affect the methylation status of ODC gene and hypomethylation may not be sufficient for TPA-increased ODC gene transcription in T24 cells.
The effects of DNA methylation on transcription and chromatin structure require that nuclear factors be able to distinguish methylated and nonmethylated DNA. We describe a methyl-CpG binding protein (MeCP) that complexes with a variety of unrelated DNA sequences when they are methylated at CpG. Fifteen or more symmetrically methylated CpG moieties per molecule are required for strong binding under our conditions. Competition experiments show that vertebrate DNAs bind to MeCP, whereas naturally nonmethylated genomes or cloned vertebrate genomes do not bind. Cross-linking experiments detect a 120 kd protein that correlates stringently with MeCP activity. Species and tissue comparisons show that MeCP is widely distributed in mammals except in embryonal carcinoma cell lines, which have very low levels.
Inactivation of normally expressed genes may play a role in the formation and/or progression of human cancers. Methylation of cytosine in DNA could potentially participate in such alterations of gene expression. Abnormalities in DNA methylation are a consistent feature of human neoplasms, and we now show that these include not only previously recognized widespread genomic hypomethylation, but also regional increases in gene methylation. A hot spot for abnormal methylation of C + G-rich areas has been detected on the short arm of chromosome 11 in an area known to harbor tumor suppressor genes. This change occurs consistently in common forms of human cancer and appears early during the transformation of cells with viruses including members of the human T-cell leukemia (HTLV) family. Furthermore, in one chromosome 11 gene examined, calcitonin, the increased methylation in somatic tumor cells coincides with the presence of an "inactive" chromatin pattern in the transcriptional regulatory area. The increased regional DNA methylation demonstrated may then participate in or mark chromosomal changes associated with gene inactivation events that are central to the genesis and/or progression of human cancers.
Evidence from many sources shows that the control of gene expression in higher organisms is related to the methylation of
cytosine in DNA, and that the pattern of methylation is inherited. Loss of methylation, which can result from DNA damage,
will lead to heritable abnormalities in gene expression, and these may be important in oncogenesis and aging. Transformed
permanent lines often lose gene activity through de novo methylation. It is proposed that epigenetic defects in germline cells
due to loss of methylation can be repaired by recombination at meiosis but that some are transmitted to offspring.
To study the mechanism of gene repression by DNA methylation, M13 gene constructs were methylated to completion and inserted into mouse L cells by DNA-mediated gene transfer. All unmethylated sequences, regardless of their source, integrated into the DNA in a potentially active DNAase I-sensitive conformation. Total CpG methylation prevented the formation of this structure and rendered these sequences DNAase I-insensitive over the entire methylated domain. Whereas unmethylated DNA demonstrated additional conformational features of active genes, such as DNAase I hypersensitivity and restriction endonuclease-sensitive segments, these markers were not present when methylated DNA was used for transfection. The use of micrococcal nuclease to probe for active or inactive supranucleosome particles also showed that DNA methylation directs DNA into an inactive type of structure. The results suggest that DNA methylation may exert its effect on gene transcription by altering both specific and nonspecific interactions between DNA and nuclear proteins.
The events occurring during emergence of cells from quiescence ("G0") are not necessarily identical to those in the G1 phase of continuously dividing cells. Cellular levels of the mRNAs coding for ornithine decarboxylase (ODC) and S-adenosyl-methionine decarboxylase (SDC), key enzymes in polyamine synthesis, increased maximally within 5 h after addition of serum to resting 3T3 cells, following a kinetic course similar to that of c-myc mRNA. In a pure early G1 population of cells, prepared by centrifugal elutriation of growing fibroblasts, the levels of ODC and SDC mRNAs were not significantly lower than in other phases of the cell cycle and approximated serum-induced levels rather than the reduced values found in serum-starved cells. Thus, we conclude that the mRNAs coding for the polyamine biosynthetic enzymes, like c-myc, are growth controlled, but not regulated during traverse of a normal cell cycle.
The organization of nucleosomes associated with a cell cycle regulated human H4 histone gene was examined in synchronized HeLa S3 cells. At various times during the cell cycle, nuclei were digested with micrococcal nuclease, and the nucleosomal pattern of the gene was obtained by Southern blot analysis using radiolabeled human histone H4 gene probes. We have detected reversible changes during the cell cycle in the chromatin structure of this gene, as reflected by the shortening of the nucleosomal spacing after replication and the peak of transcription. This variation is also observed when DNA and protein syntheses are inhibited. By using a probe that comprises 250 base pairs (bp) of the coding region and 240 bp of the 5' end of the gene, containing the promoter and DNase I sensitive sequences, we also have observed a general disruption of the nucleosomal organization, which is reflected by a degeneration of the characteristic nucleosomal ladder produced by micrococcal nuclease digestion. This modification coincides with the replication and active transcription of the gene (early S phase), which recovers its regular nucleosomal appearance when both processes have been completed, although the nucleosome linker length is shortened. When the probe utilized comprises the distal 3' end of the gene, there is no disruption of the nucleosomal pattern, but the linker region also exhibits a shortened length. A non-cell cycle regulated gene (beta-globin) does not exhibit such modifications in any of the situations analyzed.(ABSTRACT TRUNCATED AT 250 WORDS)
We have examined the expression of the transformed phenotype in a series of clonal lines of NIH/3T3 cells transfected with the human c-Ha-rasVal 12 oncogene and the neomycin phosphotransferase gene. Cells from individual transformed foci were cloned and subjected to detailed analyses of the ras sequences. Three clones were found that expressed approximately one, 2-4, or 4-8 copies of the human c-ras oncogene, respectively. A fourth clone had multiple copies of the transfected sequences, and expressed abundant c-Ha-ras RNA. Analysis of the transformed phenotype of various clones indicated that cells expressing low levels of mutant c-Ha-ras had lost some of their extracellular fibronectin network, and were barely altered in their cytoskeleton. In contrast, cells expressing abundant c-Ha-ras had lost both their actin and fibronectin networks and showed an increase in plasminogen activator activity. Cells with amplified c-Ha-rasVal 12 grew better in low serum, formed large colonies in soft agar and showed enhanced activity of ornithine decarboxylase, the rate-controlling enzyme in polyamine biosynthesis. These results show that the dosage level of the mutant oncogene makes a significant contribution to the transformed phenotype of c-Ha-ras oncogene-transformed cells.
It has been suggested that cancer represents an alteration in DNA, heritable by progeny cells, that leads to abnormally regulated expression of normal cellular genes; DNA alterations such as mutations, rearrangements and changes in methylation have been proposed to have such a role. Because of increasing evidence that DNA methylation is important in gene expression (for review see refs 7, 9-11), several investigators have studied DNA methylation in animal tumours, transformed cells and leukaemia cells in culture. The results of these studies have varied; depending on the techniques and systems used, an increase, decrease, or no change in the degree of methylation has been reported. To our knowledge, however, primary human tumour tissues have not been used in such studies. We have now examined DNA methylation in human cancer with three considerations in mind: (1) the methylation pattern of specific genes, rather than total levels of methylation, was determined; (2) human cancers and adjacent analogous normal tissues, unconditioned by culture media, were analysed; and (3) the cancers were taken from patients who had received neither radiation nor chemotherapy. In four of five patients studied, representing two histological types of cancer, substantial hypomethylation was found in genes of cancer cells compared with their normal counterparts. This hypomethylation was progressive in a metastasis from one of the patients.
In most higher organisms, DNA is modified after synthesis by the enzymatic conversion of many cytosine residues to 5-methylcytosine.
For several years, control of gene activity by DNA methylation has been recognized as a logically attractive possibility,
but experimental support has proved elusive. However, there is now reason to believe, from recent studies, that DNA methylation
is a key element in the hierarchy of control mechanisms that govern vertebrate gene function and differentiation.
We have tested the hypothesis that DNA methylation patterns are replicated in the somatic cells of vertebrates. Using M-Hpa II, the modification enzyme from Haemophilus parainfluenzae which methylates the internal cytosine residues in the sequence 5'CCGG 3' GGCC, we methylated bacteriophage phi X174 RF DNA and the cloned chicken thymidine kinase (tk) gene in vitro and then introduced these DNAs and unmethylated controls into tk- cultured mouse cells by DNA-mediated transformation. Twenty-five cell generations later, the state of methylation of transferred DNA was examined by restriction endonuclease analysis and blot hybridization. We conclude that methylation at Hpa II sites is replicated by these cultured cells but not with 100% fidelity. We have also noted that methylation of the cloned chicken tk gene decreases its apparent transformation efficiency relative to unmethylated molecules.