Discovery of TP53 splice variants in two novel papillary urothelial cancer cell lines.

BACKGROUND: Using a novel cell culture technique, we established two new cell lines, BC44 and BC61, from papillary urothelial carcinoma and analyzed them for genetic changes typical of this tumor type. METHODS AND RESULTS: Karyotyping revealed aneuploid karyotypes with loss of chromosome 9 and rearranged chromosome 5p. Molecular analysis showed CDKN2A deletions but wild-type PIK3CA. BC61 contained a G372C FGFR3 mutation. TP53 was not mutated in either cell line and BC61 expressed normal full-length protein. In contrast, BC44 exclusively expressed cytoplasmic and nuclear p53Δ40 and 133 isoforms from the alternative promoter P2 as revealed by Western blotting, immunocytochemistry and PCR. The only discernible difference in TP53 in BC44 was homozygosity for the deletion allele of the rs17878362 polymorphism in the P2 promoter. Expression of p53 isoforms was also detected in a few other urothelial carcinoma cell lines and tumor cultures and in 4 out of 28 carcinoma tissues. CONCLUSION: In urothelial cancers, TP53 is typically inactivated by mutations in one allele and loss of the wildtype allele and more frequently in invasive compared to papillary carcinomas. We show that some urothelial carcinomas may predominantly or exclusively express isoforms which are not detected by commonly used antibodies to epitopes located in the p53 TA amino-terminal region. Expression of these isoforms may constitute a further mode of p53 inactivation in urothelial carcinoma. Our findings raise the question to which extent this mechanism may compromise wildtype p53 function in papillary tumors in particular, where point mutations in the gene are rare.

Relationship of SNCG, S100A4, S100A9 and LCN2 gene expression and DNA methylation in bladder cancer.

Microarray analysis of paired cultures of normal and cancerous urothelial cells revealed differences in cytokeratin and adhesion gene expression. Normal cells expressed autocrine growth factor genes more strongly whereas carcinoma cells were distinguished by concomitant expression of urothelial and epidermal differentiation markers. Expression of SNCG, S100A9 and LCN2 was also enhanced. In other cancers, overexpression of SNCG, LCN2 and S100A4 has been ascribed to DNA hypomethylation. We therefore investigated expression and methylation of SNCG, S100A4, S100A9 and LCN2 in urothelial cancer cell lines and tissues. SNCG and S100A4 were overexpressed in some cancer tissues and cell lines, but downregulated in others, whereas LCN2 and S100A9 were upregulated in few cancer cell lines, but regularly in tissues. Normal and cancerous urothelial cells expressing SNCG lacked promoter methylation. SNCG downregulation was associated with hypermethylation and could be reversed by the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine. S100A4 methylation at regulatory intronic sites and in the promoter region was lowest in leukocytes and fibroblasts, and denser in urothelial cells. Gene expression responded to 5-aza-2'-deoxycytidine. LCN2 promoter methylation was variable and even less consistently related to expression. The S100A9 promoter was partially methylated in nonexpressing cells, but 5-aza-2'-deoxycytidine had no effect. Our data indicate that SNCG methylation is cell type-specific and the gene is hypermethylated in some urothelial cancers. S100A4, S100A9 and LCN2 are genes with moderate CpG-density that show a less stringent relationship between DNA methylation and gene expression. Therefore, changes in methylation of these genes in cancer should be interpreted cautiously.

Chromosomal instability in bladder cancer.

Chromosomal instability (CIN) distinguishes invasive urothelial carcinomas from less malignant papillary subtypes. Recent results implicate checkpoint dysfunction as a crucial factor underlying the emergence of aneuploidy in urothelial carcinogenesis. It may moreover contribute to DNA repair defects. Therefore, defects in cell cycle regulation, p53 function, and checkpoint signaling initially caused by carcinogens in the urothelium could ultimately elicit CIN. Among several mechanisms contributing to aneuploidy, breakage-fusion-bridge (BFB) cycles initiated by defective telomeres may be particularly relevant. The mechanism generating large interstitial deletions, prominently at 9p21, appears to be distinct. New experimental approaches are required to address important unresolved questions such as the precise relationship between telomere erosion and telomerase activation, the influence of checkpoint defects on DNA double-strand repair by non-homologous and homomologous recombination repair systems, and the mechanism responsible for megabase-sized interstitial deletions.

Distinctive differences in DNA double-strand break repair between normal urothelial and urothelial carcinoma cells.

Several lines of evidence suggest that defective repair of DNA double-strand breaks (DSB) contributes to genomic instability in human cancers, including urothelial carcinoma. In particular, extracts from urothelial cancers have been reported to repair DSBs preferentially by microhomology-mediated end-joining (MMEJ), considered as more error-prone than canonical non-homologous end-joining (NHEJ) predominating in normal urothelial cell extracts. However, it is not clear whether such differences are relevant to intact cells. We therefore transfected plasmids digested with different restriction enzymes to yield incompatible ends (blunt, 5'-protruding or 3'-protruding) into urothelial carcinoma cell lines or normal urothelial cells and characterized the recovered circular plasmids. All cells competently repaired DSBs in a standard cloning vector plasmid, processing 5'- as well as 3'-protruding ends. No significant differences in the extent of processing were detected and the junctions presented short microhomologies indicative of canonical NHEJ. However, dramatic and distinctive differences between normal and cancerous urothelial cells were seen in two different experiments. First, cancer cell lines processed a significantly higher fraction of plasmids cut with a single restriction enzyme that could have been repaired by direct ligation than normal cells. Secondly, for the repair of a large plasmid with incompatible ends containing a large fragment of human genomic DNA, normal cells used almost exclusively MMEJ exploiting a microhomology with the 3'-end of the break, whereas cancer cell lines often processed DNA despite suitable microhomologies. DNA repair of the small or large plasmid was almost abolished by siRNA knockdown of Ku70. These findings strongly suggest that urothelial carcinoma cells lack control mechanisms preventing overprocessing during NHEJ repair. This may account for previous findings that urothelial cancers contain unusually large chromosomal deletions. Moreover, in contrast to prevailing interpretations, our observations suggest that MMEJ, despite its error-proneness, in some instances may act as a failsafe mechanism against overprocessing during NHEJ.

Factor interaction analysis for chromosome 8 and DNA methylation alterations highlights innate immune response suppression and cytoskeletal changes in prostate cancer.

BACKGROUND: Alterations of chromosome 8 and hypomethylation of LINE-1 retrotransposons are common alterations in advanced prostate carcinoma. In a former study including many metastatic cases, they strongly correlated with each other. To elucidate a possible interaction between the two alterations, we investigated their relationship in less advanced prostate cancers. RESULTS: In 50 primary tumor tissues, no correlation was observed between chromosome 8 alterations determined by comparative genomic hybridization and LINE-1 hypomethylation measured by Southern blot hybridization. The discrepancy towards the former study, which had been dominated by advanced stage cases, suggests that both alterations converge and interact during prostate cancer progression. Therefore, interaction analysis was performed on microarray-based expression profiles of cancers harboring both alterations, only one, or none. Application of a novel bioinformatic method identified Gene Ontology (GO) groups related to innate immunity, cytoskeletal organization and cell adhesion as common targets of both alterations. Many genes targeted by their interaction were involved in type I and II interferon signaling and several were functionally related to hereditary prostate cancer genes. In addition, the interaction appeared to influence a switch in the expression pattern of EPB41L genes encoding 4.1 cytoskeleton proteins. Real-time RT-PCR revealed GADD45A, MX1, EPB41L3/DAL1, and FBLN1 as generally downregulated in prostate cancer, whereas HOXB13 and EPB41L4B were upregulated. TLR3 was downregulated in a subset of the cases and associated with recurrence. Downregulation of EPB41L3, but not of GADD45A, was associated with promoter hypermethylation, which was detected in 79% of carcinoma samples. CONCLUSION: Alterations of chromosome 8 and DNA hypomethylation in prostate cancer probably do not cause each other, but converge during progression. The present analysis implicates their interaction in innate immune response suppression and cytoskeletal changes during prostate cancer progression. The study thus highlights novel mechanisms in prostate cancer progression and identifies novel candidate genes for diagnostic and therapeutic purposes. In particular, TLR3 expression might be useful for prostate cancer prognosis and EPB41L3 hypermethylation for its detection.

In situ detection of global DNA hypomethylation in exfoliative urine cytology of patients with suspected bladder cancer.

Global DNA hypomethylation is a common phenomenon in bladder cancer. Therefore we investigated whether it is possible to detect and assess global DNA hypomethylation in bladder cancer using a specific monoclonal antibody for 5-methyl-cytosine. Cytospins from exfoliative urine cytology specimens of patients with bladder cancer or a history of bladder cancer, control patients with benign urological diseases and of young healthy volunteers were analyzed. Urothelial carcinoma (UC) cells showed various degrees of nuclear destaining indicating global DNA hypomethylation whereas all specimens from healthy volunteers showed granular nuclear staining indicating regular methylation of repeated DNA sequences. Lowest 5-methylcytosine immunostaining scores were observed in carcinoma cells and a statistically significant difference was observed between urothelial cells of healthy controls or patients with benign disease compared to bladder cancer patients (p<0.01, p<0.05, respectively). In UC cases even morphologically normal urothelial cells often displayed evident hypomethylation. Likewise, in patients with a history of UC, but no cystoscopic evidence of recurrence, morphologically non-malignant urothelial cells presented with some degree of demethylation. Our results strongly support the hypothesis of early global demethylation in bladder cancer. Immunocytochemical staining with the 5-methylcytosine antibody allows simultaneous individual assessment of nuclear morphology and methylation status of a given sample.

Expression changes in EZH2, but not in BMI-1, SIRT1, DNMT1 or DNMT3B are associated with DNA methylation changes in prostate cancer.

The polycomb proteins BMI-1, EZH2, and SIRT1 are characteristic components of the PRC1, PRC2, and PRC4 repressor complexes, respectively, that modify chromatin. Moreover, EZH2 may influence DNA methylation by direct interaction with DNA methyltransferases. EZH2 expression increases during prostate cancer progression, whereas BMI-1 and SIRT1 are not well investigated. Like EZH2 expression, DNA methylation alterations escalate in higher stage prostate cancers, raising the question whether these epigenetic changes are related. Expression of EZH2, BMI-1, SIRT1, and the DNA methyltransferases DNMT1 and DNMT3B measured by qRT-PCR in 47 primary prostate cancers was compared to APC, ASC, GSTP1, RARB2, and RASSF1A hypermethylation and LINE-1 hypomethylation. SIRT1 and DNMT3B were overexpressed in cancerous over benign tissues, whereas BMI-1 was rather downregulated and DNMT1 significantly diminished. Nevertheless, cancers with higher DNMT1 and BMI-1 expression had worse clinical characteristics, as did those with elevated EZH2. In particular, above median DNMT1 expression predicted a worse prognosis. EZH2 and SIRT1 overexpression were well correlated with increased MKI67. Immunohistochemistry confirmed limited EZH2 and heterogeneous DNMT3B overexpression and explained the decrease in BMI-1 by pronounced heterogeneity among tumor cells. EZH2 overexpression, specifically among all factors investigated, was associated with more frequent hypermethylation, in particular of GSTP1 and RARB2, and also with LINE-1 hypomethylation. Our data reveal complex changes in the composition of polycomb repressor complexes in prostate cancer. Heterogeneously expressed BMI-1 and slightly increased EZH2 may characterize less malignant cancers, whereas more aggressive cases express both at higher levels. SIRT1 appears to be generally increased in prostate cancers. Intriguingly, our data suggest a direct influence of increased EZH2 on altered DNA methylation patterns in prostate cancer.

DNA methylation alterations in urothelial carcinoma.

In urothelial cancer, hypermethylation of specific genes and genome-wide hypomethylation, reflected in decreased methylation of LINE-1 retrotransposons, have both been reported, but were never investigated in the same specimens. We analyzed hypermethylation of six genes by methylation-specific PCR and LINE-1 hypomethylation by Southern blotting in 96 carcinoma tissues. Hypermethylation frequencies were: SFRP1 (55%), APC (45%), RASSF1A (35%), DAPK1 (29%), RARB2 (19%), and CDKN2A (2%). Three groups of cancers could be discerned, with escalating hypermethylation. Hypermethylation increased with tumor stage, particularly at the transition to invasive cancers, and RARB2 hypermethylation was indicative of lymph node involvement. A comparison to a previous study on prostate cancer using the same techniques suggests that hypermethylation in urothelial carcinoma occurs in a random rather than coordinated manner. LINE-1 hypomethylation was present in 90% of specimens, largely independent of hypermethylation. Lack of hypomethylation indicated a significantly better clinical prognosis. Bisulfite sequencing of SFRP1 demonstrated dense or patchy hypermethylation in tumor tissues that likely accounts for discrepant reported frequencies. In urothelial carcinoma cell lines, the same genes as in tissues were frequently hypermethylated. SFRP1 hypermethylation was concordant with lack of expression. 5-Aza-deoxycytidine induced its reexpression in some lines, whereas additional treatment with a histone deacetylase inhibitor was required in others. Thus, epigenetic SFRP1 inactivation occurs in a graduated manner. In conclusion, markers of genome-wide hypomethylation seem optimally suited for urothelial carcinoma detection, whereas combinations of hypermethylation and hypomethylation assays hold promise for classification.

Expression of death-associated protein kinase during tumour progression of human renal cell carcinomas: hypermethylation-independent mechanisms of inactivation.

Death-associated protein kinase (DAPK) is a pro-apoptotic Ca(2+)/calmodulin-dependent serine/threonine kinase that is widely expressed in tissues but kept silent in growing cells. Downregulation of DAPK transcription by CpG methylation has been demonstrated in a variety of tumours, providing a selective growth advantage during tumour progression. As the in vivo expression of DAPK in human renal cell carcinomas (RCCs) has not previously been analysed, 72 RCCs were investigated using semi-quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). We found that almost 92% (66/72) of all primary RCCs express DAPK mRNA and results obtained from methylation-specific PCR analyses suggest that aberrant CpG methylation of the DAPK promoter is absent even in DAPK non-expressing tumours. Comparison of early/intermediate with advanced tumour stages of clear cell RCCs showed that no significant changes in the expression levels of DAPK were evident. Chromophilic/papillary RCCs display no significantly different expression patterns of DAPK compared with stage-adjusted clear cell RCCs. Furthermore, on analysing the DAPK enzyme activity in RCC cell lines with DAPK mRNA and protein expression, only 1 out of 11 cell lines showed basal DAPK activity in kinase activity assays, suggesting that DAPK, although expressed in RCC, remains largely inactive. Our study demonstrates the in vivo expression of DAPK in RCCs and reveals that, in contrast to other tumour types, RCCs may not downregulate DAPK mRNA expression during tumour progression. Despite persistent DAPK transcription and translation, however, the markedly reduced DAPK enzyme activity in our RCC cell lines suggested a post-translational inactivation of DAPK in RCCs.

Relationship of NKX3.1 and MYC gene copy number ratio and DNA hypomethylation to prostate carcinoma stage.

OBJECTIVE: High stage prostate cancers have been reported to frequently harbor chromosome 8 alterations and hypomethylation of LINE-1 retrotransposons. The potential of these parameters for molecular staging of prostate carcinoma was investigated. METHODS: High molecular weight DNA was extracted from 63 carcinoma tissues (22 pT2, 38 pT3, 3 pT4). Chromosome 8 alterations were followed by determining the ratio of NKX3.1 (at 8p21) to MYC (at 8q24) gene copy numbers (NKX3.1:MYC ratio) using a new real-time PCR technique. LINE-1 hypomethylation was quantified by Southern blot analysis. RESULTS: In 42 carcinomas NKX3.1 copy numbers were altered, with decreases in 32 cases. Copy numbers of MYC were increased in 38 cases and diminished in four. The NKX3.1:MYC ratio was altered in 45 specimens, with a decrease in all but two. NKX3.1 loss was associated with tumor stage (p<0.03) and MYC gain with Gleason score (p<0.03). The NKX3.1:MYC ratio was highly significantly associated with tumor stage (p<0.002), displaying 66% sensitivity and 87% specificity. LINE-1 hypomethylation was related (p<0.004) to tumor stage, but exhibited lower sensitivity (59%) and specificity (77%). CONCLUSION: A straightforward PCR technique detecting chromosome 8 alterations might be useful to predict which prostate cancers are organ-confined while determination of hypomethylation appears to be somewhat less well suited.

Hypomethylation of the XIST gene promoter in prostate cancer.

In a process denoted "global hypomethylation" repetitive DNA sequences like LINE-1 retrotransposons become hypomethylated in human cancers, including a subset of prostate carcinomas. It is less well known to what extent single-copy sequences are affected by this phenomenon. Therefore, we have analyzed methylation and expression of the XIST gene by bisulfite sequencing and real-time RT-PCR. The promoter of this single-copy gene is strongly methylated in normal male cells, including leukocytes and normal prostate. In prostate cancer tissues and particularly in cell lines, partial hypomethylation was observed paralleling that of LINE-1 sequences. Weak XIST expression was found in normal prostate tissues, but none in leukocytes. Only slight increases in expression of this gene were found in cancer tissues and cell lines. Our data suggest that hypomethylation in prostate cancer is indeed "global," affecting repeat and unique sequences in parallel. Detection of partially hypomethylated XIST alleles in prostate cancer tissues might be useful for the identification of cases with pronounced hypomethylation, which tend to be more aggressive.

Homozygous deletions of CDKN2A caused by alternative mechanisms in various human cancer cell lines.

The CDKN2A tumor-suppressor locus on chromosome band 9p21, which encodes p16(INK4A), a negative regulator of cyclin-dependent kinases, and p14(ARF1), an activator of TP53, is inactivated in many human cancers by point mutation, promoter hypermethylation, and, often, deletion. Homozygous deletions are unusually prevalent at this locus in very different human cancers. In the present study, we compared deletions in squamous cell carcinoma of the head and neck (SCCHN) cell lines to those in T-cell acute lymphatic leukemia (T-ALL), glioma, and bladder carcinoma (TCC) cell lines. Of 14 SCCHN lines, 10 showed homozygous deletions of CDKN2A, one displayed promoter hypermethylation with gene silencing, and one had a frameshift deletion in exon 2. Many deletion ends were in or proximal to the repetitive sequence clusters flanking the locus. Breakpoint junctions displayed variable microhomologies or insertions characteristic of DNA repair by nonhomologous end-joining. In general, deletions were much smaller in SCCHN than in TCC and glioma. In T-ALL, breakpoints were near consensus sites for recombination mediated by RAG (recombination activating genes) enzymes, and the structure of the junctions was consistent with this mechanism. We suggest that different mechanisms of CDKN2A deletion prevail in different human cancers. Aberrant RAG-mediated recombination may be responsible in T-ALL, and exuberant DNA repair by nonhomologous end-joining is the likely prevailing mechanism in SCCHN, but a distinct mechanism in TCC and glioma remains to be elucidated.

Multiple mechanisms downregulate CDKN1C in human bladder cancer.

Expression of the imprinted CDKN1C gene at chromosome 11p15.5 encoding the cell cycle inhibitor p57(KIP2) is disturbed in Beckwith-Wiedemann syndrome and in several human cancers by different mechanisms. Many advanced urothelial cancers (TCC) display downregulation of CDKN1C expression. The responsible mechanisms were investigated in TCC cell lines, with cultured normal urothelial cells (UEC) as controls. CDKN1C mRNA expression was diminished in 12/15 TCC lines and p57(KIP2) protein was decreased accordingly. Because CDKN1C is expressed from the maternal allele only, LOH at 11p15.5 represents one mechanism of downregulation. In 3 cell lines, several polymorphic markers flanking CDKN1C were homozygous compatible with this mechanism. Hypermethylation of the CDKN1C promoter, a reported cause of downregulation in other cancers, was detected by bisulfite sequencing in several cell lines and appeared associated with downregulation in at least one cell line. The methylation inhibitor 5-aza-2'deoxycytidine induced CDKN1C expression in this cell line and others. A third reported mechanism involves a switch of both alleles toward a paternal imprinting pattern, indicated by hypomethylation of a differentially methylated region (DMR) in the imprinting center (IC2). This hypomethylation was detected in most TCC lines, and was associated with re-expression of the non-coding LIT1 RNA and with downregulation of CDKN1C in several. Thus, CDKN1C downregulation in TCC seems to occur by several different mechanisms. This finding and the ability of p57(KIP2) to induce senescence in urothelial cells make CDKN1C a good candidate for a tumor suppressor at 11p in TCC.

Denaturing high-performance liquid chromatography (DHPLC) as a reliable high-throughput prescreening method for aberrant promoter methylation in cancer.

Aberrant promoter hypermethylation of CpG dinucleotides is a frequent and significant mechanism of tumor suppressor gene (TSG) silencing in cancer. As increasing numbers of downregulated putative TSGs are emerging from large-scale expression profiling studies, high-throughput techniques are needed to screen for hypermethylation. DHPLC has been established as a reliable, highly sensitive technique for mutation analysis. In this study, the use of DHPLC as a prescreening method for the identification of CpG methylation was developed by analyzing DNA samples with different, well-characterized methylation patterns of the CDKN2A/p16 promoter. Bisulfite treatment of genomic DNA was followed by PCR-amplification of unmethylated as well as methylated CDKN2A/p16 promoter sequences. PCR products were denatured and renatured, permitting the formation of heteroduplex DNA detectable by DHPLC. Methylation of all CpG-sites results in a single peak (homoduplex) with a shift in retention time, whereas partial methylation can be recognized by additional signals representing diverse heteroduplex structures. After method development, 35 DNA samples from primary bladder and breast carcinomas were analyzed in a blinded fashion, revealing complete or partial methylation of the p16 promoter in eight cases and a heterozygous mutation in one case. In conclusion, DHPLC is a highly sensitive and convenient method for methylation screening.

Correlation of hMLH1 and HPP1 hypermethylation in gastric, but not in esophageal and cardiac adenocarcinoma.

Using methylation-specific real-time PCR, we determined the prevalence of aberrant methylation in the mismatch repair gene hMLH1 and in the recently described HPP1 gene among 50 esophageal, 50 cardiac and 50 gastric ADCs. Additionally, expression of hMLH1 protein was detected immunohistochemically and correlated with DNA MSI. Hypermethylation of hMLH1 was found in 14% of esophageal, 28% of cardiac and 32% of gastric ADCs, whereas HPP1 hypermethylation was found more frequently in the 3 tumor types (64% vs. 38% vs. 54%). In gastric ADC, HPP1 hypermethylation was found more frequently in tumors with concomitant hMLH1 hypermethylation (81%) than in those without hMLH1 hypermethylation (41%, p = 0.008). Complete loss of hMLH1 protein expression, which was present in 10 carcinomas (5 cardiac and 5 gastric), was invariably correlated with hMLH1 hypermethylation and MSI. In conclusion, our data indicate that MSI and loss of the mismatch repair protein hMLH1, which is mainly caused by hMLH1 gene hypermethylation, are more prevalent in stomach and cardia carcinogenesis than in that of the esophagus. Moreover, in gastric cancer, hMLH1 hypermethylation is correlated with hypermethylation of the HPP1 gene.

DNA methylation in placentas of interspecies mouse hybrids.

Interspecific hybridization in the genus Mus results in several hybrid dysgenesis effects, such as male sterility and X-linked placental dysplasia (IHPD). The genetic or molecular basis for the placental phenotypes is at present not clear. However, an extremely complex genetic system that has been hypothesized to be caused by major epigenetic changes on the X chromosome has been shown to be active. We have investigated DNA methylation of several single genes, Atrx, Esx1, Mecp2, Pem, Psx1, Vbp1, Pou3f4, and Cdx2, and, in addition, of LINE-1 and IAP repeat sequences, in placentas and tissues of fetal day 18 mouse interspecific hybrids. Our results show some tendency toward hypomethylation in the late gestation mouse placenta. However, no differential methylation was observed in hyper- and hypoplastic hybrid placentas when compared with normal-sized littermate placentas or intraspecific Mus musculus placentas of the same developmental stage. Thus, our results strongly suggest that generalized changes in methylation patterns do not occur in trophoblast cells of such hybrids.

Peculiar structure and location of 9p21 homozygous deletion breakpoints in human cancer cells.

The CDKN2A tumor-suppressor gene in chromosome band 9p21 encoding CDKN2A (also known as p16, INK4A), a negative regulator of cyclin-dependent kinases, and p14(ARF1), an activator of TP53, is inactivated in many human cancers by point mutations, promoter hypermethylation, or deletions. Homozygous deletions predominate in certain cancer types (e.g., bladder cancers). To understand why deletions are unusually prevalent at this locus, deletions in bladder and renal cancer cell lines were mapped in detail and several deletion breakpoints cloned. Deletions were interstitial and encompassed 0.1 to >30 Mb. Most deletion breakpoints were located in or close to LINE-1 retrotransposon clusters. Therefore, deletions of CDKN2A may be facilitated by the presence of LINE-1 clusters that flank the locus. All cloned junctions were products of non-homologous recombination and consistently contained exact 2-bp microhomologies. Microhomologies are otherwise hallmarks of DNA double-strand break repair by non-homologous end joining, but the consistent size found at the CDKN2A deletion junctions is difficult to reconcile with the known properties of this process. Therefore, an unknown mechanism appears to be involved in the generation of CDKN2A deletions during carcinogenesis.

Decrease of DNA methyltransferase 1 expression relative to cell proliferation in transitional cell carcinoma.

In many common cancers such as transitional cell carcinoma (TCC), specific genes are hypermethylated, whereas overall DNA methylation is diminished. Genome-wide DNA hypomethylation mostly affects repetitive sequences such as LINE-1 retrotransposons. Methylation of these sequences depends on adequate expression of DNA methyltransferase I (DNMT1) during DNA replication. Therefore, DNMT1 expression relative to proliferation was investigated in TCC cell lines and tissue as well as in renal carcinoma (RCC) cell lines, which also display hypomethylation, as indicated by decreased LINE-1 methylation. Cultured normal uroepithelial cells or normal bladder tissue served as controls. In all tumor cell lines, DNMT1 mRNA as well as protein was decreased relative to the DNA replication factor PCNA, and DNA hypomethylation was present. However, the extents of hypomethylation and DNMT1 downregulation did not correlate. Reporter gene assays showed that the differences in DNMT1 expression between normal and tumor cells were not established at the level of DNMT1 promoter regulation. Diminished DNMT1:PCNA mRNA ratios were also found in 28/45 TCC tissues but did not correlate with the extent of DNA hypomethylation. In addition, expression of the presumed de novo methyltransferases DNMT3A and DNMT3B mRNAs was investigated. DNMT3B overexpression was observed in about half of all high-stage TCC (DNMT3B vs. tumor stage, chi(2): p = 0.03), whereas overexpression of DNMT3A was rarer and less pronounced. Expression of DNMT3A and DNMT3B in most RCC lines was higher than in TCC lines. Our data indicate that DNMT1 expression does not increase adequately with cell proliferation in bladder cancer. This relative downregulation probably contributes to hypomethylation of repetitive DNA but does not determine its extent alone.

Genomewide DNA hypomethylation is associated with alterations on chromosome 8 in prostate carcinoma.

To elucidate the relationship between genomewide DNA hypomethylation and chromosome instability, 55 prostate carcinoma specimens were analyzed for extent of hypomethylation by Southern blot analysis of LINE-1 sequence methylation and for loss or gain of chromosomal material by comparative genomic hybridization. Seventeen (31%) tumors showed strong hypomethylation of DNA, whereas four (7%) displayed slight hypomethylation and the rest of the tumors normal-level methylation. Chromosomal aberrations were observed in 34 carcinomas. The most frequent chromosomal alterations were loss of 13q in 18 cases and aberrations in 8p (loss) or 8q (gain) in 16 cases. The presence of chromosomal loss or gain was significantly associated with the presence of strong hypomethylation. A striking correlation (P = 0.00001) was observed between aberrations on chromosome 8 and hypomethylation, whereas no association was seen between DNA hypomethylation and loss of 13q. The association between DNA hypomethylation and the presence of metastases was statistically significant (P = 0.044), and both chromosomal alterations and DNA hypomethylation tended to be more frequent in higher-stage tumors. In conclusion, the data indicate that hypomethylation is associated with chromosomal instability in prostate cancer. Specifically, a surprisingly strong association between alterations on chromosome 8 and genomewide hypomethylation was found. This association suggests that DNA hypomethylation and alterations in chromosome 8 may be mechanistically linked to each other in prostate carcinoma.