H4K20me3 marks distal intergenic and repetitive regions in human mature spermatozoa.

Sperm histones represent an essential part of the paternally transmitted epigenome, but uncertainty exists about the role of those remaining in non-coding and repetitive DNA. We therefore analyzed the genome-wide distribution of the heterochromatic marker H4K20me3 in human sperm and somatic (K562) cells. To specify the function of sperm histones, we compared all H4K20me3-containing and -free loci in the sperm genome. Sperm and somatic cells possessed a very similar H4K20me3 distribution: H4K20me3 peaks occurred mostly in distal intergenic regions and repetitive gene clusters (in particular genes encoding odorant-binding factors and zinc-finger antiviral proteins). In both cell types, H4K20me3 peaks were enriched in LINEs, ERVs, satellite DNA and low complexity repeats. In contrast, H4K20me3-free nucleosomes occurred more frequently in genic regions (in particular promoters, exons, 5'-UTR and 3'-UTR) and were enriched in genes encoding developmental factors (in particular transcription activators and repressors). H4K20me3-free nucleosomes were also detected in substantial quantities in distal intergenic regions and were enriched in SINEs. Thus, evidence suggests that paternally transmitted histones may have a dual purpose: maintenance and regulation of heterochromatin and guidance towards transcription of euchromatin.

Impairment of IGF2 gene expression in prostate cancer is triggered by epigenetic dysregulation of IGF2-DMR0 and its interaction with KLF4.

BACKGROUND: Human cancer cells often exhibit impaired IGF2 expression and the underlying mechanisms are multifaceted and complex. Besides the well-known imprinting control region IGF2/H19-ICR, the involvement of a differentially methylated region in the promoter P0 of IGF2 gene (IGF2-DMR0) has been suggested. Here, we evaluate several mechanisms potentially leading to up- and/or down-regulation of IGF2 expression in prostate cancer and present a novel role of Kruppel-like factor 4 (KLF4) as a transcriptional regulator of IGF2 binding in IGF2-DMR0. METHODS: Putative binding sites for transcription factors were identified in IGF2-DMR0 using JASPAR CORE database. Gene expressions were analyzed by RT-qPCR in prostate carcinoma and adjacent benign prostate hyperplasia samples obtained by radical prostatectomy (86 RP-PCa and 47 RP-BPH) and BPH obtained by transurethral prostate resection (13 TUR-BPH). Pyrosequencing and qMSP were used for DNA methylation studies in IGF2-DMR0, IGF2/H19-ICR and Glutathione-S-transferase-P1 (GSTP1) promoter. Loss of imprinting (LOI) was analyzed by RFLP. Copy number variation (CNV) test was performed using qBiomarker CNV PCR Assay. KLF4-binding and histone-modifications were analyzed by ChIP-qPCR in prostate cancer cell lines exhibiting differentially methylated IGF2-DMR0 (LNCaP hypomethylated and DU145 hypermethylated). KLF4 protein was analyzed by western blot. Statistical associations of gene expression to methylation, IGF2 LOI and CNV were calculated by Mann-Whitney-U-test. Correlations between gene expression and methylation levels were evaluated by Spearman's-Rank-Correlation-test. RESULTS: We found a significant reduction of IGF2 expression in the majority of RP-PCa and RP-BPH in comparison to TUR-BPH. Analyzing potential molecular reasons, we found in RP-PCa and RP-BPH in comparison to TUR-BPH a significant hypomethylation of IGF2-DMR0, which coincided with hypermethylation of GSTP1-promoter, a prominent marker of prostate tumors. In contrast, IGF2 LOI and CNV did not associate significantly with up- and/or down-regulation of IGF2 expression in prostate tumors. By analyzing IGF2-DMR0, we detected a consensus sequence for KLF4 with a z-score of 7.6. Interestingly, we found that KLF4 binds to hypomethylated (17%) IGF2-DMR0 enriched with H3K9me3 and H3K27me3 (LNCaP), but does not bind under hypermethylated (85%) and H3K4me3-enriched conditions (DU145). KLF4 expression was detected in TUR-BPH as well as in RP-BPH and RP-PCa and showed a highly significant correlation to IGF2 expression. CONCLUSIONS: Our study demonstrated that in human prostate cancer the impairment of IGF2 expression is accompanied by hypomethylation of IGF2-DMR0. We revealed that KLF4 is a putative transcriptional regulator of IGF2, which binds in IGF2-DMR0 in dependence of the prevailing epigenetic state in this region. Herewith we provide complementary new insights into IGF2 dysregulation mechanisms as a critical process in prostate tumorigenesis.

Chronic Prostatitis Affects Male Reproductive Health and Is Associated with Systemic and Local Epigenetic Inactivation of C-X-C Motif Chemokine 12 Receptor C-X-C Chemokine Receptor Type 4.

Background/Aims/Objectives: Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has detrimental effects on the quality of life including the aspect of sexual dysfunction. The aim of the study was to identify if there was an adverse effect on the male genital compartment and if there are systemic or compartment-specific local signals for epigenetic dysregulation of inflammatory factors in CP/CPPS patients. METHODS: One hundred five NIH IIIb CP/CPPS patients and 41 healthy men were recruited and underwent investigations of urines, semen and blood. Promoter methylation and expression of the chemokine C-X-C motif chemokine 12 and its receptor C-X-C chemokine receptor type 4 (CXCR4) (involved in the recruitment of mast cells) were analyzed in prostate epithelial cell lines and in healthy volunteers' and patients' blood, ejaculate cell pellets, and separated ejaculate fractions (sperm and seminal somatic cells). RESULTS: Independently from age, CP/CPPS NIH IIIb was associated with significant impairment of sperm motility, morphology and semen pH (p < 0.001). Patients older than 33 years showed significantly increased seminal interleukin-8 and serum prostate specific antigen values. In patients, the CXCR4 mRNA-expression was significantly decreased in whole blood and ejaculate cell pellets due to promoter hypermethylation. Analyses on separated fractions of sperm and seminal somatic cells revealed that sperm DNA was unaffected, whereas somatic cell DNA was differentially methylated. CONCLUSIONS: NIH IIIb CP/CPPS has negative effects on surrogate parameters of male fertility and is associated significantly with systemic and local epigenetic inactivation of CXCR4.

TET enzymes are successively expressed during human spermatogenesis and their expression level is pivotal for male fertility.

STUDY QUESTION: Are ten-eleven-translocation (TET) 1-3 family enzymes involved in human spermatogenesis and do they impact male fertility? SUMMARY ANSWER: TET1, TET2 and TET3 are successively expressed at different stages of human spermatogenesis, and their expression levels associate with male fertility. WHAT IS KNOWN ALREADY: Spermatogenesis is a complex cell differentiation process accompanied by a drastic epigenetic remodeling. TET1-3 dioxygenases are essential for active DNA demethylation in the paternal pronucleus and in embryonic stem cells. STUDY DESIGN, SIZE, DURATION: Expression of TET1-3 mRNAs and proteinss and 5-hydroxymethylcytosine (5-hmC) proteins were analyzed in human testis tissues from men with obstructive azoospermia and exhibiting histologically normal spermatogenesis. Ejaculated spermatozoa from normozoospermic healthy volunteers, the 'controls' (TET1: n = 58; TET2-3: n = 63), and subfertile men who participated with their female partners in an ICSI-program, the 'patients' (TET1: n = 66; TET2-3: n = 64), were analyzed concerning the stored TET1-3 mRNAs, and the values were correlated to semen parameters and ICSI-outcomes. PARTICIPANTS/MATERIALS, SETTING, METHODS: Testis sections were used for in situ hybridization (ISH) and immunohistochemical (IHC) studies to determine TET1-3 mRNA and protein expression, and for immunofluorescence (IF) detection of 5-hmC. Sperm samples from controls were analyzed by western blot, immunocytochemistry (ICC) and RT-PCR concerning the presence of non-degraded TET1-3 protein and mRNA. Sperm samples from controls and patients were used for quantitative TET1-3 mRNA analyses (reverse transcription-polymerase chain reaction) and for comparative statistical evaluations under consideration of semen parameters and ICSI-outcome (pregnancy). MAIN RESULTS AND THE ROLE OF CHANCE: During human spermatogenesis TET1-3 proteins are successively expressed: TET2 is expressed in the cytoplasm of late pachytene spermatocytes of Stage V, TET1 starts to be expressed in the nuclei of Step 1 round spermatids at Stage I, and TET3 starts to be expressed in the nuclei of Step 3 round spermatids at Stage III. Five-hmC appears only in Step 5 elongated spermatids. All three TETs are still detectable at the mRNA and protein level in sperm cells in considerable amounts. Control men generally exhibited higher levels of TET1-3 in sperm. TET1- and TET3-mRNA levels in sperm were significantly negatively correlated with age (P = 0.0025 and P = 0.0343) and positively correlated with progressive sperm motility (P = 0.0007 and P = 0.018). All TETs showed a significant association with sperm concentration (P < 0.03). Patients diagnosed with oligozoospermia and/or asthenozoospermia (TET1: n = 35; TET2-3: n = 32) showed significantly reduced TET1-3 in sperm in comparison to controls (P = 0.003, P = 0.041 and P = 0.028), but not compared with normozoospermic patients. Levels of TET3 in sperm was significantly associated with high-fertilization rates (P = 0.009). Concerning ICSI-outcome, the lowest levels of TET1-3 mRNAs in sperm were found in the non-pregnant group. Increased TET2 in sperm was significantly associated with pregnancy (P = 0.006). LIMITATIONS, REASONS FOR CAUTION: Our results concerning the association of the mRNA level of TETs in ejaculated sperm cells to different fertility parameters are descriptive. Further studies clarifying the reasons for decreased TET1-3 levels in subfertile men and their effect on their sperm methylome are essential. WIDER IMPLICATIONS OF THE FINDINGS: The study gives a substantial indication that in human spermiogenesis, an active DNA demethylation process occurs with an involvement of TET enzymes, and that the level of TET1-3 expression is pivotal for male fertility. STUDY FUNDING: Research grant from the German Research Foundation (DFG) to U.S. (SCHA1531/1-1 and SCHA1531/2-1). COMPETING INTERESTS: None.

The sperm protamine mRNA ratio as a clinical parameter to estimate the fertilizing potential of men taking part in an ART programme.

STUDY QUESTION: Could the protamine-1 to protamine-2 mRNA ratio serve as a biomarker to estimate the fertilizing capacity of sperm from men taking part in an IVF/ICSI programme? SUMMARY ANSWER: The protamine mRNA ratio clearly discriminates between fertile and subfertile men and sperm with a normal protamine mRNA ratio exhibit a higher fertilizing capacity in IVF/ICSI. WHAT IS KNOWN ALREADY: Aberrant sperm protamine ratios are associated with male factor infertility and mRNA ratio is comparable with protein ratio (due to transcriptional stop in elongating spermatids). STUDY DESIGN, SIZE, DURATION: The study population was drawn from subfertile men, whose female partners participated in IVF or ICSI programmes between September 2010 and February 2012. Normozoospermic healthy volunteers served as controls. Sperm cells were lysed, mRNA extracted, reverse transcribed and subjected to real-time quantitative PCR using specific primer pairs for protamine-1 and protamine-2. Relative protamine-1 and protamine-2 mRNA levels were analysed with the Mann-Whitney U-test (two-tailed). PARTICIPANTS/MATERIALS, SETTING, METHODS: Quantitative RT-PCR for protamines 1 and 2 has been performed in ejaculates from 32 normozoospermic volunteers (control, University Clinic Giessen, Germany) and 306 patients, whose female partners took part in an IVF (n = 76; University Clinic Hamburg, Germany and Shanghai Jiaotong University, China) or an ICSI (n = 230; University Clinic Munich, Germany and Kinderwunschzentrum Wiesbaden, Germany) programme. MAIN RESULTS AND THE ROLE OF CHANCE: The sperm protamine mRNA ratio in normozoospermic men (0.98 ± 0.3) differed significantly from that of ICSI patients (Munich 0.81 ± 0.1; Wiesbaden 0.78 ± 0.2; P < 0.001), while processed samples obtained from IVF patients revealed a normal protamine mRNA ratio (Hamburg 1.0 ± 0.07; Shanghai 1.0 ± 0.54). Normal protamine mRNA ratios were associated with a significantly higher total motile sperm count and a significantly higher percentage of progressively motile sperm. Sperm with a normal protamine mRNA ratio revealed a higher fertilization capacity (fc) in both IVF (53.6% of patients with fc > 80%; P = 0.017) and ICSI (65.1% of patients with fc > 70%; P = 0.028). LIMITATIONS, REASONS FOR CAUTION: The protamine mRNA ratio in an individual sperm cell used for ICSI may be different from the overall value obtained from a semen aliquot. WIDER IMPLICATIONS OF THE FINDINGS: Data are in line with current literature and suggest the protamine mRNA ratio as a diagnostic marker to estimate the fertilizing capacity of sperm. STUDY FUNDING: The German Research Foundation (DFG) to K.S., W.W. and A.P. (STE 892/9-2), as well as to A.S. and H.C.O. (SP721/1-3). COMPETING INTEREST(S): None.

Epigenetic down regulation of RASSF10 and its possible clinical implication in prostate carcinoma.

BACKGROUND: Ras association domain family (RASSF) comprises several tumor suppressor genes, which are often epigenetically inactivated in human tumors. Here, we aim to analyze the relevance of the recently identified member RASSF10 in prostate carcinogenesis. METHODS: RASSF10 promoter methylation and mRNA expression were investigated by bisulfite-pyrosequencing and qRT-PCR, respectively, in prostate carcinoma (PCa) cell lines (LNCaP, 22Rv1, DU-145) and in 83 primary PCa and 53 primary benign prostatic hyperplasia (BPH) tissues obtained after radical prostatectomy. Histological localization of RASSF10 was done by in situ hybridization. To prove the epigenetic nature of RASSF10 down regulation, PCa cell lines were treated with 5-aza-2-deoxycytidine and trichostatin A. Potential function of RASSF10 was analyzed in LNCaP by colony formation and apoptosis assays. RESULTS: RASSF10 mRNA was localized to cells of the basal layer of the prostatic gland. Absence (LNCaP) and decrease (22Rv1, DU-145) of RASSF10 expression was associated with promoter methylation and could be restored by demethylating agents. A link between RASSF10 mRNA reduction and promoter methylation was also detected in primary prostate tissues (P = 0.006), where PCa showed more frequently reduced RASSF10 levels when compared with BPH (33.7% vs. 13.2%, P = 0.009). RASSF10 methylation could be further associated with advanced tumor stage and advanced age (P-values < 0.05). Our preliminary functional assays revealed the ability of RASSF10 to inhibit colony formation (P = 0.018) and to increase apoptosis (P = 0.035). CONCLUSIONS: This is the first study, which demonstrates the frequent epigenetic inactivation of RASSF10 in PCa and its implication in clinical symptoms of PCa.

Identification of 5 novel genes methylated in breast and other epithelial cancers.

BACKGROUND: There are several high throughput approaches to identify methylated genes in cancer. We utilized one such recently developed approach, MIRA (methylated-CpG island recovery assay) combined with CpG island arrays to identify novel genes that are epigenetically inactivated in breast cancer. RESULTS: Using this approach we identified numerous CpG islands that demonstrated aberrant DNA methylation in breast cancer cell lines. Using a combination of COBRA and sequencing of bisulphite modified DNA, we confirmed 5 novel genes frequently methylated in breast tumours; EMILIN2, SALL1, DBC1, FBLN2 and CIDE-A. Methylation frequencies ranged from between 25% and 63% in primary breast tumours, whilst matched normal breast tissue DNA was either unmethylated or demonstrated a much lower frequency of methylation compared to malignant breast tissue DNA. Furthermore expression of the above 5 genes was shown to be restored following treatment with a demethylating agent in methylated breast cancer cell lines. We have expanded this analysis across three other common epithelial cancers (lung, colorectal, prostate). We demonstrate that the above genes show varying levels of methylation in these cancers. Lastly and most importantly methylation of EMILIN2 was associated with poorer clinical outcome in breast cancer and was strongly associated with estrogen receptor as well as progesterone receptor positive breast cancers. CONCLUSION: The combination of the MIRA assay with CpG island arrays is a very useful technique for identifying epigenetically inactivated genes in cancer genomes and can provide molecular markers for early cancer diagnosis, prognosis and epigenetic therapy.

Elevated seminal plasma estradiol and epigenetic inactivation of ESR1 and ESR2 is associated with CP/CPPS.

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is associated with urinary tract symptoms and hormonal imbalances amongst others. The heterogeneous clinical presentation, unexplored molecular background and lack of prostate biopsies complicate therapy. Here, using liquid biopsies, we performed a comprehensive translational study on men diagnosed with CP/CPPS type III (n= 50; median age 39.8, range 23-65) and age-matched controls (n= 61; median age 36.8, range 20-69), considering biochemical parameters of blood and ejaculates, and epigenetic regulation of the estrogen receptor genes (ESR1 and ESR2) in leukocytes isolated from blood (systemic regulation) and in somatic cells isolated from ejaculates (local regulation). We found elevated 17ß-estradiol (E2) levels in seminal plasma, but not in blood plasma, that was significantly associated with CP/CPPS and impaired urinary tract symptoms. In ejaculated somatic cells of CP/CPPS patients we found that ESR1 and ESR2 were both significantly higher methylated in CpG-promoters and expressionally down-regulated in comparison to controls. Mast cells are reported to contribute to CP/CPPS and are estrogen responsive. Consistent with this, we found that E2 -treatment of human mast cell lines (HMC-1 and LAD2) resulted in altered cytokine and chemokine expression. Interestingly, in HMC-1 cells, possessing epigenetically inactivated ESR1 and ESR2, E2 -treatment led to a reduced transcription of a number of inflammatory genes. Overall, these data suggest that elevated local E2 levels associate with an epigenetic down-regulation of the estrogen receptors and have a prominent role in CP/CPPS. Investigating E2 levels in semen could therefore serve as a promising biomarker to select patients for estrogen targeted therapy.

E-cadherin and DAP kinase in pancreatic adenocarcinoma and corresponding lymph node metastases.

E-cadherin and DAP kinase have been implicated as 'invasion suppressor' genes in human cancer. The aim of this study was to analyze the methylation status of E-cadherin and DAP kinase and the expression of the protein in the metastatic lesions and to compare it with the expression in the primary tumor. Methylation-specific PCR of the DAP kinase and E-cadherin promoter was performed in 28 primary adenocarcinomas of the pancreas and in 13 corresponding regional lymph node metastases. The presence of E-cadherin and DAP kinase protein was assessed by immunohistochemistry. Metastatic lymph nodes showed a significant different expression profile from the primary tumor. E-cadherin methylation was observed in 8/28 (29%) and loss of protein expression was observed in 16/28 (57%) of pancreatic carcinomas. E-cadherin methylation was observed in 7/13 (54%) and loss of protein expression was observed in 11/13 (85%) lymph node metastases (p=0.047). DAP kinase methylation occurred in 11/28 (39%) pancreatic carcinomas and loss of protein expression was observed in 13/28 (46%). DAP kinase was methylated in 6/13 (46%) lymph node meta-stases and loss of protein expression was observed in 10/13 (77%) (p=0.039). Comparing primary tumor and corresponding lymph node metastases in 13 cases, the status of E-cadherin methylation was discordant in 2 cases. The protein expression pattern of E-cadherin and DAP kinase was discordant in 4 and 3 cases respectively. Unmethylated tumor samples did not express E-cadherin in 12 and DAP kinase protein in 6 cases. Our results demonstrate that reduction of E-cadherin and DAP kinase expression is more frequent in lymph node metastases than in the primary tumor and methylation of the promoter region contributes to this reduction; however, an alternative mechanism of inactivation seems to exist.

Uniformity of nucleosome preservation pattern in Mammalian sperm and its connection to repetitive DNA elements.

Nucleosome-to-protamine exchange during mammalian spermiogenesis is essential for compaction and protection of paternal DNA. It is interesting that, depending on the species, 1% to 15% of nucleosomes are retained, but the generalizability and biological function of this retention are unknown. Here, we show concordantly in human and bovine that nucleosomes remained in sperm chromatin predominantly within distal intergenic regions and introns and associated with centromere repeats and retrotransposons (LINE1 and SINEs). In contrast, nucleosome depletion concerned particularly exons, 5'-UTR, 3'-UTR, TSS, and TTS and was associated with simple and low-complexity repeats. Overlap of human and bovine genes exhibiting nucleosome preservation in the promoter and gene body revealed a significant enrichment of signal transduction and RNA- and protein-processing factors. Our study demonstrates the genome-wide uniformity of the nucleosome preservation pattern in mammalian sperm and its connection to repetitive DNA elements and suggests a function in preimplantation processes for paternally derived nucleosomes.

Frequent aberrant methylation of the imprinted IGF2/H19 locus and LINE1 hypomethylation in ovarian carcinoma.

Epigenetic alteration of tumor-related genes through changes of DNA methylation is a hallmark for carcinogenesis and aberrant DNA methylation modulates the activity of tumor suppressor genes, imprinted genes and repetitive elements. In ovarian carcinoma, frequent loss of imprinting or aberrant methylation of repetitive elements were reported, however, combined analysis were not performed. We analyzed the aberrant methylation of a differentially methylated region (DMR0) and a CTCF binding site of the IGF2-H19 locus and methylation of LINE1 and Satellite 2 in 22 primary ovarian carcinomas (OC) and controls by a quantitative bisulfite restriction analysis (QUBRA). In 91% of OC, a significant hypomethylation of DMR0 was found compared to controls (p<0.05). In 77% of OC, a hypermethylation of a CTCF binding site was found (p<0.05). A combined hypomethylation of DMR0 and hypermethylation of the CTCF binding was observed in 73% of OC. Hypomethylation of LINE1 and Satellite 2 was detected in 100 and 23% of OC, respectively. In summary, we found frequent combined aberrant methylation of the IGF2-H19 locus and LINE1 in the vast majority of OC, suggesting that these changes are important events in tumorigenesis.

Modulation of actin filament dynamics by actin-binding proteins residing in lamellipodia.

Lamellipodial extension depends essentially on the polymerisation cycle of actin. In this cellular compartment the rate and extent of actin polymerisation is tightly regulated by a large number of actin-binding proteins. The main regulators comprise proteins of the actin-depolymerising factor (ADF)/cofilin family, which stimulate actin cycling, but there are also minor constituents like gelsolin and certain variants of tropomyosin that have so far not been considered to be lamellipodial constituents. A number of cell lines express ADF and cofilin simultaneously as shown here for the fibroblastic normal rat kidney (NRK) cell line. Both proteins co-localise in the lamellipodial region. We furthermore demonstrate the presence of gelsolin in lamellipodia by immunostaining with anti-gelsolin antibodies and transfection with EGFP-tagged gelsolin constructs. The presence of tropomyosins in lamellipodia has recently been reported (Hillberg et al., 2006. Tropomyosins are present in lamellipodia of motile cells. Eur. J. Cell Biol. 85, 399-409). In order to evaluate the effect of the simultaneous presence of ADF and cofilin together with tropomyosin and/or gelsolin on the polymerisation cycle of actin, we analysed their effect or combinations of these actin-binding proteins on the steady-state F-actin-ATPase activity in biochemical assays. Our results demonstrate stimulatory effects of ADF/cofilin on actin cycling and a further modulation of ADF/cofilin-stimulated F-actin-ATPase activity by gelsolin and tropomyosin in a complex manner.

The role of promoter CpG methylation in the epigenetic control of stem cell related genes during differentiation.

Differentiation and malignant transformation of stem cells are regulated by epigenetic mechanisms. We analyzed promoter methylation and expression of the stem cell determining genes Brachyury, DPPA5, FGF4, FOXD3, LIN28, NESTIN and ZFP42 depending on the differentiation state in human mesenchymal stem cells (MSC), human embryonal carcinoma cells (ECC) and somatic tumor cells. Differentiation of MSC into osteoblasts and adipocytes was accompanied with a loss of expression of the Brachyury gene and downregulation of LIN28. Inactivation of Brachyury was associated with progressive methylation of its CpG island promoter. In ECC promoter methylation of stem cell markers was more frequent in the differentiated subgroup (71%) compared to undifferentiated ECC (29%) and this was associated with downregulation of Brachyury, DPPA5, FGF4, FOXD3, LIN28 and ZFP42. DPPA5 was methylated and NESTIN was unmethylated in most tumor cells. In somatic tumor cells, methylation of stem cell markers (Brachyury, DPPA5, FGF4, FOXD3, LIN28 and ZFP42) was frequently observed (85%). Treatment of cell lines with an inhibitor of DNA methyltransferase reactivated the expression of DPPA5, FGF4, FOXD3, LIN28 and ZFP42, indicating that aberrant promoter methylation is a crucial event that results in their silencing. Our results suggest that epigenetic inactivation of stem cell associated genes is mediated by promoter methylation and that this may represent a fundamental mechanism during normal differentiation processes.

TNP-470 fails to block the onset of angiogenesis and early tumor establishment in an intravital minimal disease model.

BACKGROUND AND AIMS: The angiogenesis inhibitor TNP-470 (AGM-1470) has shown encouraging results in animal models of established tumors. However, results of recent clinical trials using TNP-470 have been disappointing. Since little is known about the effects of TNP-470 at the minimal disease stage, we analyzed the effects of TNP-470 on the early stages of tumor establishment. METHODS: Twenty thousand green fluorescent protein (GFP)-transfected murine CT-26 (colonic carcinoma) or Panc-02-H0 (pancreatic adenocarcinoma) cells were inoculated in dorsal skin-fold chambers in BALB/c or C57BL6 mice. Tumor area and microvessel density (MVD) were quantified by intravital microscopy (IVM). Body weight was also monitored. Effects were compared with those in a conventional model involving subcutaneous (s.c.) inoculation of 10(6) tumor cells, followed by measurement of tumor volume, endogenous plasma VEGF/endostatin (ELISA) and proliferation/apoptosis/microvessel density (Ki-67/TUNEL/CD-34). TNP-470 was injected s.c. over the 10-day experimental period (30 mg/kg every 2 days [n=6] to 100 mg/kg/day [n=5 dorsal skin-fold chamber model, n=4 s.c. tumor model]). RESULTS: At 30 mg/kg/every second day neither CT-26 nor PANC-02-H0 tumors were inhibited in neither of the two models. TNP-470 dosage was escalated in CT-26-bearing animals until an antiangiogenic effect could be observed. In the IVM model, only TNP-470 100 mg/kg/day reduced MVD (P=0.006), but failed to block the onset of angiogenesis and tumor area increase. Body weight decreased by 25% (P<0.05). In the subcutaneous tumor model, tumor growth was reduced (P=0.045) but not blocked, while vascular endothelial growth factor (VEGF)/endostatin synthesis and Ki67/TUNEL/CD-34 were not significantly affected. CONCLUSION: While capable of reducing tumor growth in a conventional model, treatment with TNP-470 does not block the onset of angiogenesis and tumor establishment in a model of minimal disease. When used as a single agent TNP-470 does not control minimal tumor disease in experimental colonic carcinoma.

Overexpression of cyclin E protein is closely related to the mutator phenotype of colorectal carcinoma.

BACKGROUND AND AIMS: A subset of colorectal carcinomas are due to a deficiency in the DNA mismatch repair system. The molecular mechanisms of tumorigenesis in these tumors is not yet well understood. Deregulation of the cell cycle, specifically of the G(1) and S phases, is a hallmark of human cancers. Transition from the G(1) to the S phase is accelerated by increased cyclin E protein expression, and recent studies suggest that overexpression of cyclin E leads to chromosomal instability. The overexpression of cyclin E in a variety of human cancers, for example in colorectal, gastric, lung, breast, and kidney cancer, provides evidence that cyclin E plays a pivotal role in the cell cycle and replication. We examined whether the overexpression of cyclin E is related to the status of the mismatch repair system in colorectal carcinomas. PATIENTS AND METHODS: Frozen tumor samples and adjacent normal colon mucosa obtained from 100 patients were subjected to microsatellite analysis, RT-PCR, western blot analysis and immunohistochemistry. RESULTS: High microsatellite instability was detected in 13 tumors, and in 10 of these (77%) cyclin E protein was overexpressed at least twofold compared to normal mucosa. In contrast, only 28 of the remaining 87 microsatellite stable tumors (32%) overexpressed cyclin E. Lower molecular weight cyclin E proteins were present in 7 of 87 microsatellite stable carcinoma (8%), compared to 7 cases exhibiting lower molecular weight isoforms of 13 MSI carcinoma (54%). CONCLUSION: Increased cyclin E protein expression and the appearance of lower molecular weight cyclin E proteins were significantly associated with MSI in colorectal tumors. The data indicate that increased and/or aberrant expression of cyclin E protein might contribute to the mutator phenotype of colorectal cancer.