Methylation of endogenous human retroelements in health and disease.

Retroelements constitute approximately 45% of the human genome. Long interspersed nuclear element (LINE) autonomous retrotransposons are predominantly represented by LINE-1, nonautonomous small interspersed nuclear elements (SINEs) are primarily represented by ALUs, and LTR retrotransposons by several families of human endogenous retroviruses (HERVs). The vast majority of LINE and HERV elements are densely methylated in normal somatic cells and contained in inactive chromatin. Methylation and chromatin structure together ensure a stable equilibrium between retroelements and their host. Hypomethylation and expression in developing germ cells opens a "window of opportunity" for retrotransposition and recombination that contribute to human evolution, but also inherited disease. In somatic cells, the presence of retroelements may be exploited to organize the genome into active and inactive regions, to separate domains and functional regions within one chromatin domain, to suppress transcriptional noise, and to regulate transcript stability. Retroelements, particularly ALUs, may also fulfill physiological roles during responses to stress and infections. Reactivation and hypomethylation of LINEs and HERVs may be important in the pathophysiology of cancer and various autoimmune diseases, contributing to chromosomal instability and chronically aberrant immune responses. The emerging insights into the pathophysiological importance of endogenous retroelements accentuate the gaps in our knowledge of how these elements are controlled in normal developing and mature cells.

Coordinate hypermethylation at specific genes in prostate carcinoma precedes LINE-1 hypomethylation.

In prostate carcinoma (PCa) increased DNA methylation ('hypermethylation') occurs at specific genes such as GSTP1. Nevertheless, overall methylation can be decreased ('hypomethylation') because methylation of repetitive sequences like LINE-1 retrotransposons is diminished. We analysed DNA from 113 PCa and 36 noncancerous prostate tissues for LINE-1 hypomethylation by a sensitive Southern technique and for hypermethylation at eight loci by methylation-specific PCR. Hypermethylation frequencies for GSTP1, RARB2, RASSF1A, and APC in carcinoma tissues were each >70%, strongly correlating with each other (P<10(-6)). Hypermethylation at each locus was significantly different between tumour and normal tissues (10(-11)82% of PCas. PCa may fall into three classes, that is, with few DNA methylation changes, with frequent hypermethylation, or with additional LINE-1 hypomethylation.

Transcriptional regulation of the human LINE-1 retrotransposon L1.2B.

Although LINE-1 (L1) sequences constitute the most important family of retrotransposons in the human genome, their transcriptional regulation is poorly understood. Specifically, their unusual internal promoter is incompletely characterized. Current promoter prediction programs fail to identify the promoter in the 5'UTR of the active LINE-1 element L1.2B. Experimental investigation of this promoter using reporter gene assays in various human and murine cell types confirmed that the promoter consists of two segments, and demonstrated that the distal portion is essential for cell-type-independent activity. No differences in promoter activity were found between normal and transformed cells. The complete promoter was shown to possess approximately 20% of the activity of the strong early promoter of cytomegalovirus, and to be capable of directing the expression of levels of p53 sufficient to kill normal and transformed human cells. Thus, active LINE-1 elements contain highly active promoters capable of driving cell-type-independent expression, which are of potential use in mammalian expression constructs. In vitro methylation of the promoter at HpaII sites decreased its activity independently of cell type, but this repression was alleviated in MBD2-/- cells. Surprisingly, mutation of specific HpaII sites was also found to reduce promoter activity. Thus, efficient repression of the L1.2B promoter by DNA methylation may involve MBD2 binding, but at least one HpaII site also appears to be involved specifically in transcriptional activation. Since neither promoter activity nor the efficiency of repression by methylation differed between normal and tumor cells, the re-activation of LINE-1 sequences observed in tumor cells is probably caused by hypomethylation of the promoter.

Polymorphic methyl group metabolism genes in patients with transitional cell carcinoma of the urinary bladder.

Because polymorphisms in the methyl group metabolism genes methylene-tetrahydrofolate reductase (MTHFR), methionine synthase (MS), and cystathione beta-synthetase (CBS) affect plasma homocysteine levels and intracellular concentrations of S-adenosylmethionine (SAM), they modify the susceptibility to cardiovascular diseases and cancer. Specifically, genome-wide decreased DNA methylation ('hypomethylation') in human cancers might be a consequence of decreased SAM levels. Because hypomethylation is particularly prevalent in transitional cell carcinoma of the urinary bladder (TCC), the genotype distributions for the two each most prevalent MTHFR, MS, and CBS alleles were compared between 165 TCC patients and 150 population controls. The distributions of the MTHFR 677A/V and the MS 919G/D alleles were not significantly different between cancer patients and controls, even after stratification according to age, gender, tumor stage or grade. The CBS 844INS68 allele was slightly less frequent in TCC patients than in controls (q=0.07 versus 0.10), but was rarer among males in both groups. Among the TCC patients, this gender difference was highly significant (Mantel-Haenszel and chi(2)-test P=0.007). No significant difference between TCC patients and controls was found for any combined genotype. Likewise, the extent of DNA hypomethylation determined in 62 carcinoma specimens was not related to the respective genotypes. Thus, on their own, the MTHFR, MS and CBS genotypes do not appear to act upon susceptibility to TCC or influence the extent of DNA hypomethylation in this cancer.

Up-regulation of cyclin-dependent kinase 4/cyclin D2 expression but down-regulation of cyclin-dependent kinase 2/cyclin E in testicular germ cell tumors.

Testicular germ cell tumors (GCT) characteristically display two chromosome 12 abnormalities: the isochromosome i(12p) and concomitant deletions of the long arm. Some genes important in the control of the G(1)-S cell cycle checkpoint G(1)-S, i.e., cyclin-dependent kinases 2 and 4, cyclin D2 are located on this chromosomal region. Therefore, testicular GCTs were analyzed as to the expression of CDK2, CDK4, CDK6, and the expression of their catalytic partners cyclins D1, D2 and E by semiquantitative reverse transcription-PCR. Cyclin D2, located on 12p, was overexpressed in 69% (31 of 45) of the tumors by a mean factor of 8, including all histological subtypes. In addition, the cyclin D2 partner CDK4 was increased in 41% (21 of 51) of all tumors by a factor of 6, most strongly in embryonal carcinomas. Sixty-four percent of the seminomas and 23% of the non-seminomas had decreased expression of CDK6 by a mean factor of 5 (P = 0.009). Statistical analysis using configural frequency analysis and regression analysis revealed that cyclin D2 and CDK4 expression were strongly correlated (r(2) = 0.682; P = 0.000052), whereas expression of CDK6 did not correlate with either of them (r(2) = 0.382; P = 0.00085). CDK2 and its catalytic partner cyclin E were down-regulated in 40% (19 of 47) and 42% (19 of 45) of the tumors, respectively, by a factor of 7 each. Western blots and immunohistochemical experiments confirmed cyclin D2 and CDK4 overrepresentation and reduced expression of cyclin E and CDK2 tumors in the few tumors under protein study. Despite its localization on 12q13, a hot spot for loss of heterozygosity in testicular GCTs (>40%), Southern blotting revealed no gross DNA alteration of the CDK2 gene. Because up-regulation of the cyclin D2/CDK4 complex and down-regulation of cyclin E/CDK2 complex were found in seminomas as well as in non-seminomas and in all tumor stages, these findings seem to be early events during tumorigenesis of testicular GCTS: Together with previous findings that retinoblastoma mRNA and protein expression is strongly decreased in these tumors, these data suggest an unusual deregulated G(1)-S checkpoint as a decisive event for germ cell tumors.

Glutathione transferase isozyme genotypes in patients with prostate and bladder carcinoma.

Genotype distributions for GSTP1, GSTM1, and GSTT1 were determined in 91 patients with prostatic carcinoma and 135 patients with bladder carcinoma and compared with those in 127 abdominal surgery patients without malignancies. None of the genotypes differed significantly with respect to age or sex among controls or cancer patients. In the group of prostatic carcinoma patients, GSTT1 null allele homozygotes were more prevalent (25% in carcinoma patients vs. 13% in controls, Fisher P =0.02, chi2 P=0.02, OR=2.31, CI = 1.17-4.59) and the combined M1-/T1 -null genotype was also more frequent (9% vs. 3%, chi2 P=0.02, Fisher P = 0.03). Homozygosity for the GSTM1 null allele was more frequent among bladder carcinoma patients (59% in bladder carcinoma patients vs 45% in controls, Fisher P=0.03, chi2 P=0.02, OR=1.76, CI=1.08-2.88). In contrast to a previous report, no significant increase in the frequency of the GSTP1b allele was found in the tumor patients. Except for the combined GSTM1-/ T1-null genotype in prostatic carcinoma, none of the combined genotypes showed a significant association with either of the cancers. These findings suggest that specific single polymorphic GST genes, that is GSTM1 in the case of bladder cancer and GSTT1 in the case of prostatic carcinoma, are most relevant for the development of these urological malignancies among the general population in Central Europe.

Methyl group metabolism gene polymorphisms and susceptibility to prostatic carcinoma.

BACKGROUND: Alterations of DNA methylation are very frequent in prostatic carcinoma. A possible cause underlying altered DNA methylation could be an insufficient level of S-adenosylmethionine as a consequence of nutritional imbalances or of weaker alleles of genes for its synthesis, i.e., encoding methylene-tetrahydrofolate reductase (MTHFR), methionine synthase (MS), and beta-cystathione synthetase (CBS). Therefore, homozygosity or heterozygosity for such weaker alleles may underlie susceptibility to prostatic carcinoma. METHODS: The distribution of the two most frequent MTHFR, MS, and CBS alleles was determined in 132 prostatic carcinoma patients and 150 population controls by restriction fragment length polymorphism-(RFLP) PCR. RESULTS: In the controls, a Hardy-Weinberg equilibrium was observed for each allele pair. No significant differences were observed with respect to age or gender. No significant differences for single genes or combinations were found between prostatic carcinoma patients and controls, although the MTHFR Val allele was slightly overrepresented among the tumor patients. Neither did the allele distribution significantly differ among the prostatic carcinoma patients stratified according to age, clinical stage, or presence of metastases. However, the MTHFR Val allele tended to be associated with higher tumor grade. CONCLUSIONS: In general, the data do not support the hypothesis that weaker alleles in methyl group metabolism genes constitute a major factor in the high prevalence of DNA methylation alterations found in prostatic carcinoma. However, a potential association with the MTHFR genotype deserves further study.

Cyclin-dependent kinase inhibitor P27(KIP1) is expressed preferentially in early stages of urothelial carcinoma.

OBJECTIVES: To evaluate the expression of p27(KIP1) and p21(CIP1) and the prognostic values of both markers in urothelial carcinoma. The expression of the cyclin-dependent kinase inhibitor p27(KIP1) characterizes early-stage and well-differentiated carcinomas of the colon, breast, and prostate and is associated with an improved prognosis. In urothelial carcinoma, its expression has not been as well investigated. Another cyclin-dependent kinase inhibitor, p21(CIP1), is expressed in early-stage bladder tumors, but published data on its prognostic value are contradictory. METHODS: Expression of p27(KIP1) and p21(CIP1) was analyzed by immunohistochemistry in 114 urothelial carcinoma specimens from 77 patients. The Ki67 index was determined as an indicator of cell proliferation. The expression of the markers was correlated with tumor recurrence and progression during an average follow-up period of 3.9 years. RESULTS: Expression of p27(KIP1) was significantly more frequent in superficial than in muscle-invasive tumors (chi-square test, P = 0.012; Fisher's exact test, P = 0.014). Although similar overall, the expression pattern of p21(CIP1) did not match on a tumor-by-tumor basis. No correlation was seen with the Ki67 index. Patients with tumors displaying strong positive staining for p27(KIP1) or p21(CIP1) had fewer recurrences and progression events, but the difference was not statistically significant. Instead, a Ki67 index of less than 10% was significantly (P = 0.0335) related to a lack of recurrence. CONCLUSIONS: Neither p27(KIP1) nor p21(CIP1) appear to be good predictors of tumor progression in urothelial carcinoma, even though their expression is strongly decreased in muscle-invasive tumors.