Galactic Cosmic Radiation Induces Persistent Epigenome Alterations Relevant to Human Lung Cancer.

Human deep space and planetary travel is limited by uncertainties regarding the health risks associated with exposure to galactic cosmic radiation (GCR), and in particular the high linear energy transfer (LET), heavy ion component. Here we assessed the impact of two high-LET ions 56Fe and 28Si, and low-LET X rays on genome-wide methylation patterns in human bronchial epithelial cells. We found that all three radiation types induced rapid and stable changes in DNA methylation but at distinct subsets of CpG sites affecting different chromatin compartments. The 56Fe ions induced mostly hypermethylation, and primarily affected sites in open chromatin regions including enhancers, promoters and the edges ("shores") of CpG islands. The 28Si ion-exposure had mixed effects, inducing both hyper and hypomethylation and affecting sites in more repressed heterochromatic environments, whereas X rays induced mostly hypomethylation, primarily at sites in gene bodies and intergenic regions. Significantly, the methylation status of 56Fe ion sensitive sites, but not those affected by X ray or 28Si ions, discriminated tumor from normal tissue for human lung adenocarcinomas and squamous cell carcinomas. Thus, high-LET radiation exposure leaves a lasting imprint on the epigenome, and affects sites relevant to human lung cancer. These methylation signatures may prove useful in monitoring the cumulative biological impact and associated cancer risks encountered by astronauts in deep space.

Dual role of TMS1/ASC in death receptor signaling.

Aberrant DNA methylation of promoter region CpG islands is associated with gene silencing and serves as an alternative to mutations in the inactivation of tumor suppressor genes in human cancers. We identified a gene TMS1 (for Target of Methylation-mediated Silencing) that is subject to such epigenetic silencing in a significant proportion of human breast and other cancers. Also known as ASC and PYCARD, TMS1 encodes a bipartite intracellular signaling molecule with proposed roles in apoptosis and inflammation. However, the precise role of this protein in the pathogenesis of breast and other cancers has not been clearly defined. In this study, we examined the role of TMS1/ASC in death receptor signaling. We found that TMS1/ASC is upregulated in response to treatment with TNF-related apoptosis-inducing ligand (TRAIL) and tumor necrosis factor-alpha (TNFalpha) in breast epithelial cells, but not in human fibroblasts. This upregulation was not dependent on the synthesis of a TNFalpha-regulated intermediate or alterations in mRNA stability, suggesting a direct effect on TMS1/ASC transcription. Induction of TMS1/ASC by TNFalpha was blocked by co-expression of a dominant negative IkappaBalpha, small interfering RNA-mediated knockdown of RelA/p65, or concurrent treatment with SP600125, indicating a requirement for the nuclear factor-kappaB (NF-kappaB) and jun kinase signaling pathways. Although previous work has suggested that TMS1/ASC may be directly regulated by p53, we found that whereas treatment of breast epithelial cells or normal diploid fibroblasts with DNA damaging agents resulted in the stabilization of endogenous p53 and a concomitant increase in p21, it had little impact on the expression of TMS1/ASC mRNA or protein. We further show that whereas TMS1/ASC is not required for TNFalpha or TRAIL-induced activation of NF-kappaB or caspase-8, it can promote caspase-8 activation independently of death receptor-ligand interactions. Taken together, these data suggest that upregulation of TMS1/ASC by TNFalpha and subsequent activation of caspase-8 could function to amplify the apoptotic signal induced by death receptors in some cell types, including breast epithelial cells.

Leptin promotes the proliferative response and invasiveness in human endometrial cancer cells by activating multiple signal-transduction pathways.

An increase in the risk of cancer is one of the consequences of obesity. The predominant cancers associated with obesity have a hormonal basis and include breast, prostate, endometrium, colon and gall-bladder cancers. Leptin, the key player in the regulation of energy balance and body weight control also acts as a growth factor on certain organs in both normal and disease states. Therefore, it is plausible that leptin acts to promote cancer growth by acting as a mitogenic agent. However, a direct role for leptin in endometrial cancer has not been demonstrated. In this study, we analyzed the proliferative role of leptin and the mechanism(s) underlying this action in endometrial cancers which express both short and long isoforms of leptin receptors. Treatment with leptin resulted in increased proliferation of ECC1 and Ishikawa cells. The promotion of endometrial cancer cell proliferation by leptin involves activation of STAT3 and ERK2 signaling pathways. Moreover, leptin-induced phosphorylation of ERK2 and AKT was dependent on JAK/STAT activation. Therefore blocking its action at the JAK/STAT level could be a rational therapeutic strategy for endometrial carcinoma in obese patients. We also found that leptin potently induces invasion of endometrial cancer cells in a Matrigel invasion assay. Leptin-stimulated invasion was effectively blocked by pharmacological inhibitors of JAK/STAT (AG490) and phosphatidylinositol 3-kinase (LY294002). Taken together these data indicate that leptin promotes endometrial cancer growth and invasiveness and implicate the JAK/STAT and AKT pathways as critical mediators of leptin action. Our findings have potential clinical implications for endometrial cancer progression in obese patients.

TMS1/ASC: the cancer connection.

TMS1/ASC is a bipartite protein comprising two protein-protein interaction domains, a pyrin domain (PYD) and a caspase recruitment domain (CARD). Proteins containing these domains play pivotal roles in regulating apoptosis and immune response pathways, and mutations in a number of PYD- and CARD-containing proteins have been linked to autoinflammatory diseases and cancer. Indeed, one of the ways in which TMS1/ASC was identified was as a target of methylation-mediated silencing in breast cancer cells. This review discusses the mounting evidence supporting a correlation between the silencing of TMS1/ASC expression and cancer. In addition, it addresses the reported functions of TMS1/ASC that include apoptosis, activation of inflammatory caspases and regulation of NF-kappa B, and discusses the potential ways in which loss of TMS1/ASC contributes to carcinogenesis.

Predicting aberrant CpG island methylation.

Epigenetic silencing associated with aberrant methylation of promoter region CpG islands is one mechanism leading to loss of tumor suppressor function in human cancer. Profiling of CpG island methylation indicates that some genes are more frequently methylated than others, and that each tumor type is associated with a unique set of methylated genes. However, little is known about why certain genes succumb to this aberrant event. To address this question, we used Restriction Landmark Genome Scanning to analyze the susceptibility of 1,749 unselected CpG islands to de novo methylation driven by overexpression of DNA cytosine-5-methyltransferase 1 (DNMT1). We found that although the overall incidence of CpG island methylation was increased in cells overexpressing DNMT1, not all loci were equally affected. The majority of CpG islands (69.9%) were resistant to de novo methylation, regardless of DNMT1 overexpression. In contrast, we identified a subset of methylation-prone CpG islands (3.8%) that were consistently hypermethylated in multiple DNMT1 overexpressing clones. Methylation-prone and methylation-resistant CpG islands were not significantly different with respect to size, C+G content, CpG frequency, chromosomal location, or promoter association. We used DNA pattern recognition and supervised learning techniques to derive a classification function based on the frequency of seven novel sequence patterns that was capable of discriminating methylation-prone from methylation-resistant CpG islands with 82% accuracy. The data indicate that CpG islands differ in their intrinsic susceptibility to de novo methylation, and suggest that the propensity for a CpG island to become aberrantly methylated can be predicted based on its sequence context.

Identification and characterization of the human MOG1 gene.

Many of the proteins that mediate transport into and out of the nucleus have been structurally and functionally conserved throughout evolution. Here we describe the sequence and characterization of the human MOG1 gene. The MOG1 gene was originally identified in Saccharomyces cerevisiae as a multi-copy suppressor of conditional alleles of the yeast nuclear transport factor, GSP1 (scRan) (Oki and Nishimoto (1998) Proc. Natl. Acad. Sci. USA 95, 15388-15393). A search of the expressed sequence tag database identified a putative human protein that is 29% identical and 47% similar to the yeast protein. Our experiments demonstrate that the human MOG1 message is expressed in a variety of tissue samples. Several experiments indicate that the human MOG1 protein binds to both yeast and human Ran suggesting functional conservation between the yeast and human MOG1 proteins. Furthermore, hMOG1a, like scMOG1, is localized throughout the cell but is concentrated within the nucleus. Consistent with these findings, hMOG1a can partially complement the growth defect present in yeast MOG1 deletion cells. Taken together, our findings suggest that MOG1 is an evolutionarily conserved Ran binding protein that could play a role in regulating nuclear protein trafficking.

Activation of a caspase-9-mediated apoptotic pathway by subcellular redistribution of the novel caspase recruitment domain protein TMS1.

Genetic and epigenetic alterations affecting proteins involved in apoptosis can contribute to the establishment and progression of cancer. Recently, our laboratory has isolated a novel gene, TMS1, that is aberrantly methylated and silenced in a significant proportion of human breast cancers. TMS1 contains a caspase recruitment domain (CARD), suggesting a role in caspase-mediated cell death. In the present study, we characterize the participation of TMS1 in apoptosis and examine the subcellular localization of the protein. Inducible expression of TMS1 inhibited cellular proliferation and induced DNA fragmentation in a time-dependent manner. These apoptotic events were blocked by the general caspase inhibitor, Z-VAD-fmk. The ability of TMS1 to trigger apoptosis was also suppressed by a dominant negative form of caspase-9 but not by a dominant negative form of caspase-8, indicating that TMS1 functions through activation of caspase-9. Unlike a number of other CARD-containing proteins, TMS1 did not activate nuclear factor kappaB-dependent transcription, consistent with a proapoptotic role for TMS1 in death signaling pathways. Timed localization studies revealed that TMS1-induced apoptosis was accompanied by the redistribution of TMS1 from the cytoplasm to perinuclear spherical structures. Whereas the apoptotic activity of TMS1 was blocked by caspase inhibition, the formation of TMS1-containing subcellular structures was not, suggesting that the redistribution of TMS1 precedes caspase activation. Both the proapoptotic activity of TMS1 and aggregate formation were dependent on the CARD. In summary, the data indicate that TMS1-induced apoptosis proceeds through a CARD-dependent aggregation step followed by activation of a caspase-9-mediated pathway.

TMS1, a novel proapoptotic caspase recruitment domain protein, is a target of methylation-induced gene silencing in human breast cancers.

Gene silencing associated with aberrant methylation of promoter region CpG islands is an acquired epigenetic alteration that serves as an alternative to genetic defects in the inactivation of tumor suppressor and other genes in human cancers. The hypothesis that aberrant methylation plays a direct causal role in carcinogenesis hinges on the question of whether aberrant methylation is sufficient to drive gene silencing. To identify downstream targets of methylation-induced gene silencing, we used a human cell model in which aberrant CpG island methylation is induced by ectopic expression of DNA methyltransferase. Here we report the isolation and characterization of TMS1 (target of methylation-induced silencing), a novel CpG island-associated gene that becomes hypermethylated and silenced in cells overexpressing DNA cytosine-5-methyltransferase-1. We also show that TMS1 is aberrantly methylated and silenced in human breast cancer cells. Forty percent (11 of 27) of primary breast tumors exhibited aberrant methylation of TMS1. TMS1 is localized to chromosome 16p11.2-12.1 and encodes a 22-kDa predicted protein containing a COOH-terminal caspase recruitment domain, a recently described protein interaction motif found in apoptotic signaling molecules. Ectopic expression of TMS1 induced apoptosis in 293 cells and inhibited the survival of human breast cancer cells. The data suggest that methylation-mediated silencing of TMS1 confers a survival advantage by allowing cells to escape from apoptosis, supporting a new role for aberrant methylation in breast tumorigenesis.

Alterations in DNA methylation: a fundamental aspect of neoplasia.

Neoplastic cells simultaneously harbor widespread genomic hypomethylation, more regional areas of hypermethylation, and increased DNA-methyltransferase (DNA-MTase) activity. Each component of this "methylation imbalance" may fundamentally contribute to tumor progression. The precise role of the hypomethylation is unclear, but this change may well be involved in the widespread chromosomal alterations in tumor cells. A main target of the regional hypermethylation are normally unmethylated CpG islands located in gene promoter regions. This hypermethylation correlates with transcriptional repression that can serve as an alternative to coding region mutations for inactivation of tumor suppressor genes, including p16, p15, VHL, and E-cad. Each gene can be partially reactivated by demethylation, and the selective advantage for loss of gene function is identical to that seen for loss by classic mutations. How abnormal methylation, in general, and hypermethylation, in particular, evolve during tumorigenesis are just beginning to be defined. Normally, unmethylated CpG islands appear protected from dense methylation affecting immediate flanking regions. In neoplastic cells, this protection is lost, possibly by chronic exposure to increased DNA-MTase activity and/or disruption of local protective mechanisms. Hypermethylation of some genes appears to occur only after onset of neoplastic evolution, whereas others, including the estrogen receptor, become hypermethylated in normal cells during aging. This latter change may predispose to neoplasia because tumors frequently are hypermethylated for these same genes. A model is proposed wherein tumor progression results from episodic clonal expansion of heterogeneous cell populations driven by continuous interaction between these methylation abnormalities and classic genetic changes.

Mapping patterns of CpG island methylation in normal and neoplastic cells implicates both upstream and downstream regions in de novo methylation.

Promoter region CpG island methylation is associated with tumor suppressor gene silencing in neoplasia. GenBank sequence analyses revealed that a number of CpG islands are juxtaposed to multiple Alu repeats, which have been proposed as "de novo methylation centers." These islands also contain multiple Sp1 elements located upstream and downstream of transcription start, which have been shown to protect CpG islands from methylation. We mapped the methylation patterns of the E-cadherin (E-cad) and von Hippel-Lindau (VHL) tumor suppressor gene CpG island regions in normal and neoplastic cells. Although unmethylated in normal tissue, these islands were embedded between densely methylated flanking regions containing multiple Alu repeats. These methylated flanks were segregated from the unmethylated, island CpG sites by Sp1-rich boundary regions. Finally, in human fibroblasts overexpressing DNA methyltransferase, de novo methylation of the E-cad CpG island initially involved sequences at both ends of the island and the adjacent, flanking regions and progressed with time to encompass the entire CpG island region. Together, these data suggest that boundaries exist at both ends of a CpG island to maintain the unmethylated state in normal tissue and that these boundaries may be progressively overridden, eliciting the de novo methylation associated with tumor suppressor gene silencing in neoplasia.

Role of estrogen receptor gene demethylation and DNA methyltransferase.DNA adduct formation in 5-aza-2'deoxycytidine-induced cytotoxicity in human breast cancer cells.

The cytosine analog 5-aza-2'-deoxycytidine is a potent inhibitor of DNA methyltransferase. Its cytotoxicity has been attributed to several possible mechanisms including reexpression of growth suppressor genes and formation of covalent adducts between DNA methyltransferase and 5-aza-2'-deoxycytidine-substituted DNA which may lead to steric inhibition of DNA function. In this study, we use a panel of human breast cancer cell lines as a model system to examine the relative contribution of two mechanisms, gene reactivation and adduct formation. Estrogen receptor-negative cells, which have a hypermethylated estrogen receptor gene promoter, are more sensitive than estrogen receptor-positive cells and underwent apoptosis in response to 5-aza-2'-deoxycytidine. For the first time, we show that reactivation of a gene silenced by methylation, estrogen receptor, plays a major role in this toxicity in one estrogen receptor-negative cell line as treatment of the cells with anti-estrogen-blocked cell death. However, drug sensitivity of other tumor cell lines correlated best with increased levels of DNA methyltransferase activity and formation DNA.DNA methyltransferase adducts as analyzed in situ. Therefore, both reexpression of genes like estrogen receptor and formation of covalent enzyme. DNA adducts can play a role in 5-aza-2'-deoxycytidine toxicity in cancer cells.

New 5' regions of the murine and human genes for DNA (cytosine-5)-methyltransferase.

DNA (cytosine-5)-methyltransferases (EC 2.1.1.37) maintain patterns of methylated cytosine residues in the mammalian genome; faithful maintenance of methylation patterns is required for normal development of mice, and aberrant methylation patterns are associated with certain human tumors and developmental abnormalities. The organization of coding sequences at the 5'-end of the murine and human DNA methyltransferase genes was investigated, and the DNA methyltransferase open reading frame was found to be longer than previously suspected. Expression of the complete open reading frame by in vitro transcription-translation and by transfection of expression constructs into COS7 cells resulted in the production of an active DNA methyltransferase of the same apparent mass as the endogenous protein, while translation from the second in-frame ATG codon produced a slightly smaller but fully active protein. Characterization of mRNA 5' sequences and the intron-exon structure of the 5' region of the murine and human genes indicated that a previously described promoter element (Rouleau, J., Tanigawa, G., and Szyf, M. (1992) J. Biol. Chem. 267, 7368-7377) actually lies in an intron that is more than 5 kilobases downstream of the transcription start sites.

Switch from monoallelic to biallelic human IGF2 promoter methylation during aging and carcinogenesis.

We have previously linked aging, carcinogenesis, and de novo methylation within the promoter of the estrogen receptor (ER) gene in human colon. We now examine the dynamics of this process for the imprinted gene for insulin-like growth factor II (IGF2). In young individuals, the P2-4 promoters of IGF2 are methylated exclusively on the silenced maternal allele. During aging, this promoter methylation becomes more extensive and involves the originally unmethylated allele. Most adult human tumors, including colon, breast, lung, and leukemias, exhibit increased methylation at the P2-4 IGF2 promoters, suggesting further spreading during the neoplastic process. In tumors, this methylation is associated with diminished or absent IGF2 expression from the methylated P3 promoter but maintained expression from P1, an upstream promoter that is not contained within the IGF2 CpG island. Our results demonstrate a remarkable evolution of methylation patterns in the imprinted promoter of the IGF2 gene during aging and carcinogenesis, and provide further evidence for a potential link between aberrant methylation and diseases of aging.

De novo methylation of CpG island sequences in human fibroblasts overexpressing DNA (cytosine-5-)-methyltransferase.

Recent studies showing a correlation between the levels of DNA (cytosine-5-)-methyltransferase (DNA MTase) enzyme activity and tumorigenicity have implicated this enzyme in the carcinogenic process. Moreover, hypermethylation of CpG island-containing promoters is associated with the inactivation of genes important to tumor initiation and progression. One proposed role for DNA MTase in tumorigenesis is therefore a direct role in the de novo methylation of these otherwise unmethylated CpG islands. In this study, we sought to determine whether increased levels of DNA MTase could directly affect CpG island methylation. A full-length cDNA for human DNA MTase driven by the cytomegalovirus promoter was constitutively expressed in human fibroblasts. Individual clones derived from cells transfected with DNA MTase (HMT) expressed 1- to 50-fold the level of DNA MTase protein and enzyme activity of the parental cell line or clones transfected with the control vector alone (Neo). To determine the effects of DNA MTase overexpression on CpG island methylation, we examined 12 endogenous CpG island loci in the HMT clones. HMT clones expressing > or = 9-fold the parental levels of DNA MTase activity were significantly hypermethylated relative to at least 11 Neo clones at five CpG island loci. In the HMT clones, methylation reached nearly 100% at susceptible CpG island loci with time in culture. In contrast, there was little change in the methylation status in the Neo clones over the same time frame. Taken together, the data indicate that overexpression of DNA MTase can drive the de novo methylation of susceptible CpG island loci, thus providing support for the idea that DNA MTase can contribute to tumor progression through CpG island methylation-mediated gene inactivation.

Stabilization of DNA methyltransferase levels and CpG island hypermethylation precede SV40-induced immortalization of human fibroblasts.

De novo methylation of normally unmethylated CpG islands and increased expression of DNA (cytosine-5)-methyltransferase (DNA MTase) are common characteristics of immortalized cell lines and human tumors. To examine the acquisition of these properties with respect to cellular immortalization, we studied CpG island methylation and DNA MTase expression in aging normal human fibroblasts and their SV40-infected preimmortal (precrisis) and immortal (postcrisis) derivatives. The levels of DNA MTase enzyme activity decreased by 50% as normal fibroblasts were cultured to senescence. By contrast, DNA MTase activity did not decrease in SV40-infected pre- or postcrisis cells but remained similar to that of young fibroblasts and 2-4-fold higher than that of senescent fibroblasts. DNA MTase mRNA levels paralleled those of enzyme activity. Several loci were examined to determine the relationship between the dynamics of DNA MTase expression and the appearance of de novo CpG island methylation. Ten CpG island loci examined were unmethylated in normal young fibroblasts. By contrast, four of these loci (the CALC1, MyoD, and IGF-2 genes on chromosome 11p and the estrogen receptor gene on chromosome 6q) were de novo methylated in fully immortalized, postcrisis cells. Two of these four were actually methylated in extended life span precrisis cells, and one, the estrogen receptor locus, exhibited de novo methylation with aging in normal fibroblasts. The data indicate that an ability to maintain DNA MTase levels is acquired with SV40-induced escape from senescence. Furthermore, aberrant CpG island methylation can be established prior to immortalization, either as a function of normal aging or in response to SV40-induced escape from senescence.

Increased cytosine DNA-methyltransferase activity during colon cancer progression.

BACKGROUND: Molecular changes during progressive stages of colon cancer and other human tumors commonly involve altered regulation of DNA methylation. These changes include overall genomic hypomethylation, regional hypermethylation, and increased levels of messenger RNA (mRNA) for cytosine DNA-methyltransferase (DNA-MTase), the enzyme that catalyzes DNA methylation at CpG (cytosine-phospho-guanine) sites. This increase in DNA-MTase transcripts (mRNA), if accompanied by increased DNA-MTase activity, could play a role in the abnormal DNA methylation patterns that appear early in colon tumor progression. PURPOSE: We sought to determine whether increased DNA-MTase mRNA levels during colon cancer progression are associated with increased cellular DNA-MTase enzymatic activity. METHODS: We adapted a microassay for DNA-MTase and used it to measure activity in human colon carcinoma and in colon mucosa of normal control subjects and of patients with colon cancer or with familial adenomatous polyposis (FAP), which is a risk factor for colon cancer. Steady-state DNA-MTase gene transcripts were measured by a reverse transcriptase polymerase chain reaction assay. To compare DNA-MTase activity with mRNA levels, we determined both variables simultaneously for one colon cancer specimen, its adjacent mucosa, and the colon mucosa of a control patient and compared the values. RESULTS: Compared with DNA-MTase activity in mucosa from normal control subjects, activity was elevated 1.4-fold in FAP mucosa, 1.6-fold in the uninvolved mucosa of patients with cancer, and 5.4-fold in the cancer specimens. All these differences were statistically significant. Fourteen of 15 cancer samples and 47% of the uninvolved adjacent mucosa samples had values that were higher than the highest value in normal mucosa. In one patient who had both a benign adenomatous polyp and a malignant adenocarcinoma, increasing DNA-MTase activity was observed at each stage of tumor progression. CONCLUSION: These results demonstrate that an increased DNA methylation capacity accompanies the increase in DNA-MTase transcripts observed during progressive stages of colon cancer. IMPLICATION: Further studies are needed to determine whether this abnormal methylation capacity plays a role in establishing the abnormal DNA methylation patterns seen in human malignancies.

Altered chromosomal methylation patterns accompany oncogene-induced transformation of human bronchial epithelial cells.

Abnormal methylation of CpG island sequences on chromosomes 11p and 17p, and tumor phenotype-associated differential methylation of chromosome 3p loci have been described in human lung tumors (S.B. Baylin, J.W.M. Hoppener, A. de Bustros, P.H. Steenbergh, C.J.M. Lips, and B. D. Nelkin, Cancer Res., 46: 2917-2922, 1986; M. Makos, B.D. Nelkin, M. I. Lerman, F. Latif, B. Zbar, and S.B. Baylin, Proc. Natl. Acad. Sci. USA, 89: 1929-1933, 1992; A. de Bustros, B. D. Nelkin, A. Silverman, G. Ehrlich, B. Poiesz, and S. B. Baylin, Proc. Natl. Acad. Sci. USA, 85: 5693-5697, 1988). Using an in vitro model of lung tumor progression, we now show that these aberrant methylation patterns occur at different stages during cellular immortalization and oncogene-induced neoplastic transformation of normal human bronchial epithelial cells (NHBE). The CALC1 CpG island locus on chromosome 11p15.4 was essentially unmethylated in NHBE and simian virus 40 T-antigen immortalized NHBE (BEAS-2B cells) but became de novo methylated in 5 of 6 BEAS-2B derived cell lines that were transfected or infected with various oncogenes and in a spontaneously neoplastically transformed subline of BEAS-2B cells. By contrast, an additional CpG island locus, pYNZ22, at 17p13.3 became fully methylated following the immortalization of NHBE and was not further changed by oncogene-induced transformation. Finally, at a non-CpG island locus pYNZ86.1 on chromosome 3p14, different tumor phenotype-associated methylation patterns became apparent only after passage of the turmorigenic oncogene-transformed bronchial epithelial cell lines in athymic nude mice. Whereas cell lines derived from tumors with a non-small cell lung carcinoma-like phenotype were significantly hypomethylated relative to their parental cell lines, a cell line derived from a tumor with a more small cell lung carcinoma-like phenotype retained the methylation status of its parental cell line. The data indicate that altered DNA methylation patterns, including the de novo methylation of normally unmethylated CpG island sequences and demethylation of nonisland sequences, arise at different stages during immortalization and oncogene-induced neoplastic transformation of bronchial epithelial cells. These findings suggest that DNA methylation abnormalities accompany, or may play a role in, the genetic changes that occur during lung tumor progression.

Isolation and characterization of the cDNA encoding human DNA methyltransferase.

We have cloned a series of overlapping cDNA clones encoding a 5194 bp transcript for human DNA methyltransferase (DNA MTase). This sequence potentially codes for a protein of 1495 amino acids with a predicted molecular weight of 169 kDa. The human DNA MTase cDNA has eighty percent homology at the nucleotide level, and the predicted protein has seventy-four percent identity at the amino acid level, to the DNA MTase cDNA cloned from mouse cells. Like the murine DNA MTase, the amino terminal two-thirds of the human protein contains a cysteine-rich region suggestive of a metal-binding domain. The carboxy terminal one-third of the protein shows considerable similarity to prokaryotic (cytosine-5)-methyltransferases. The arrangement of multiple motifs conserved in the prokaryotic genes is preserved in the human DNA MTase, including the relative position of a proline-cysteine dipeptide thought to be an essential catalytic site in all (cytosine-5)-methyltransferases. A single 5.2 kb transcript was detected in all human tissues tested, with the highest levels of expression observed in RNA from placenta, brain, heart and lung. DNA MTase cDNA clones were used to screen a chromosome 19 genomic cosmid library. The DNA MTase-positive cosmids which are estimated to span a genomic distance of 93 kb have been localized to 19p13.2-p13.3 by fluorescence in situ hybridization. Isolation of the cDNA for human DNA MTase will allow further study of the regulation of DNA MTase expression, and of the role of this enzyme in establishing DNA methylation patterns in both normal and neoplastic cells.

Selective cellular depletion of mitochondrial DNA by the polyamine analog N1,N12-bis(ethyl)spermine and its relationship to polyamine structure and function.

N1,N8-Bis(ethyl)spermidine (BESPD) and N1,N12-bis(ethyl)spermine (BESPM) are minimally modified analogs of spermidine and spermine that deplete cellular polyamine pools by suppressing key polyamine biosynthetic enzymes. The consequences of polyamine depletion and the concomitant analog replacement of these pools were compared on two cellular DNA targets, mitochondrial DNA (mtDNA) and a defined nuclear DNA episome present in 935.1 mouse fibroblasts. The spermidine analog, BESPD, depleted cellular putrescine and spermidine pools, but not spermine pools, and had no effect on either DNA target. Treatment with the corresponding analog of spermine, BESPM, resulted in a near-total depletion of all three polyamine pools and a greater than 80% reduction in the cellular content of mtDNA, without affecting the levels of the nuclear episome. Topological forms analysis by Southern blotting of mtDNA and episomal DNA from BESPM-treated cells failed to reveal any forms interconversion, indicating the absence of analog-induced single- or double-strand break damage to either DNA target. The growth-dependent loss of mtDNA is consistent with a rapid cessation of mtDNA replication and subsequent dilution of existing mtDNA copies by cell division. Similar decreases in polyamine pools and mtDNA were also observed in L1210 cells treated with BESPM. When a comparable level of polyamine depletion was produced in L1210 cells by specific enzyme inhibitors, there was no effect on the cellular content of mtDNA, and BESPD was not rendered capable of decreasing mtDNA levels. Because the analogs are structurally similar to the naturally occurring polyamines and would be expected to have similar binding properties, the loss in mtDNA may reflect dysfunctional replacement by BESPM at spermine-specific binding sites in the mitochondrion.