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Biomarkers with relevance for loco-regional therapy are needed in human papillomavirus negative aka HPV(-) head and neck squamous cell carcinoma (HNSCC). Based on the premise that DNA methylation pattern is highly conserved, we sought to develop a reliable and robust methylome-based classifier identifying HPV(-) HNSCC patients at risk for loco-regional recurrence (LR) and all-event progression after postoperative radiochemotherapy (PORT-C). The training cohort consisted of HPV-DNA negative HNSCC patients (n = 128) homogeneously treated with PORT-C in frame of the German Cancer Consortium-Radiation Oncology Group (DKTK-ROG) multicenter biomarker trial. DNA Methylation analysis was performed using Illumina 450 K and 850 K-EPIC microarray technology. The performance of the classifier was integrated with a series of biomarkers studied in the training set namely hypoxia-, 5-microRNA (5-miR), stem-cell gene-expression signatures and immunohistochemistry (IHC)-based immunological characterization of tumors (CD3/CD8/PD-L1/PD1). Validation occurred in an independent cohort of HPV(-) HNSCC patients, pooled from two German centers (n = 125). We identified a 38-methylation probe-based HPV(-) Independent Classifier of disease Recurrence (HICR) with high prognostic value for LR, distant metastasis and overall survival (P < 10 -9 ). HICR remained significant after multivariate analysis adjusting for anatomical site, lymph node extracapsular extension (ECE) and size (T-stage). HICR high-risk tumors were enriched for younger patients with hypoxic tumors (15-gene signature) and elevated 5-miR score. After adjustment for hypoxia and 5-miR covariates, HICR maintained predicting all endpoints. HICR provides a novel mean for assessing the risk of LR in HPV(-) HNSCC patients treated with PORT-C and opens a new opportunity for biomarker-assisted stratification and therapy adaptation in these patients., (© 2021 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

PARP1 inhibitors (PARPis) are used clinically during cancer therapy and are thought to exert their cytotoxicity through PARP1 polymerase inhibition and PARP1-DNA trapping. Here, we showed no significant correlation between PARP1-DNA trapping and cytotoxicity induced by PARPis. We complemented PARP1-knockout sublines with wild-type PARP1 and 11 mutants with different point mutations that affect the polymerase activity. When examining the PARPi talazoparib, the induced cytotoxicity was highly significantly correlated with cellular PARP1 polymerase activity, but not with its PARP1-DNA trapping or polymerase inhibition. Similarly, talazoparib's PARP1-DNA trapping revealed significant correlation with the polymerase activity rather than its inhibition. Differently, however, when evaluating purified wild-type and mutated PARP1, we identified an almost linear relationship between PARPis' inhibiting PARP1 dissociation from DNA and their cytotoxicity in 17 cancer cell lines. In contrast, no significant correlation existed between PARP1 polymerase inhibition in the histone-based systems and the cytotoxicity. After careful comparisons on different methods and detection targets, we conclude that the PARPi-mediated increase in PARP1-DNA binding by inhibiting autoPARylation of PARP1 on DNA rather than in PARP1-DNA trapping is correlated with PARPi's cytotoxicity. Accordingly, we established a new PARPi screening model that more closely predicts cytotoxicity., (© 2019 UICC.)

Colonic and rectal cancers differ in their clinicopathologic features and treatment strategies. Molecular markers such as gene methylation, microsatellite instability and KRAS mutations, are becoming increasingly important in guiding treatment decisions in colorectal cancer. However, their association with clinicopathologic variables and utility in the management of rectal cancer is still poorly understood. We analyzed CDKN2A gene methylation, CpG island methylator phenotype (CIMP), microsatellite instability and KRAS/BRAF mutations in a cohort of 381 rectal cancers with extensive clinical follow-up data. BRAF mutations (2%), CIMP-high (4%) and microsatellite instability-high (2%) were rare, whereas KRAS mutations (39%), CDKN2A methylation (20%) and CIMP-low (25%) were more common. Only CDKN2A methylation and KRAS mutations showed an association with poor overall survival but these did not remain significant when analyzed with other clinicopathologic factors. In contrast, this prognostic effect was strengthened by the joint presence of CDKN2A methylation and KRAS mutations, which independently predicted recurrence of cancer and was associated with poor overall and cancer-specific survival. This study has identified a subgroup of more aggressive rectal cancers that may arise through the KRAS-p16 pathway. It has been previously shown that an interaction of p16 deficiency and oncogenic KRAS promotes carcinogenesis in the mouse and is characterized by loss of oncogene-induced senescence. These findings may provide avenues for the discovery of new treatments in rectal cancer., (© 2013 UICC.)

Greater exposure to estrogens is a risk factor for ovarian cancer. To investigate the role of estrogens in ovarian cancer, a spot urine sample and a saliva sample were obtained from 33 women with ovarian cancer and 34 age-matched controls. Thirty-eight estrogen metabolites, conjugates and DNA adducts were analyzed in the urine samples using ultraperformance liquid chromatography/tandem mass spectrometry, and the ratio of adducts to metabolites and conjugates was calculated for each sample. The ratio of depurinating estrogen-DNA adducts to estrogen metabolites and conjugates was significantly higher in cases compared to controls (p < 0.0001), demonstrating high specificity and sensitivity. DNA was purified from the saliva samples and analyzed for genetic polymorphisms in the genes for two estrogen-metabolizing enzymes. Women with two low-activity alleles of catechol-O-methyltransferase plus one or two high-activity alleles of cytochrome P450 1B1 had higher levels of estrogen-DNA adducts and were more likely to have ovarian cancer. These findings indicate that estrogen metabolism is unbalanced in ovarian cancer and suggest that formation of estrogen-DNA adducts plays a critical role in the initiation of ovarian cancer., (© 2013 UICC.)

Aristolochic acid (AA), a component of all Aristolochia-based herbal medicines, is a potent nephrotoxin and human carcinogen associated with upper urinary tract urothelial carcinoma (UUC). To investigate the clinical and pathological characteristics of AA-induced UUC, this study included 152 UUC patients, 93 of whom had been exposed to AA based on the presence of aristolactam-DNA adducts in the renal cortex. Gene sequencing was used to identify tumors with A:T-to-T:A transversions in TP53, a mutational signature associated with AA. Cases with both aristolactam-DNA adducts and A:T-to-T:A transversions in TP53 were defined as AA-UUC, whereas patients lacking both of these biomarkers were classified as non-AA-UUC. Cases with either biomarker were classified as possible-AA-UUC. Forty (26%), 60 (40%), and 52 (34%) patients were classified as AA-UUC, possible-AA-UUC and non-AA-UUC, respectively. AA-UUC patients were younger (median ages: 64, 68, 68 years, respectively; p=0.189), predominately female (65%, 42%, 35%, respectively; p=0.011), had more end-stage renal disease (28%, 10%, 12%, respectively; p=0.055), and were infrequent smokers (5%, 22%, 33%, respectively; p=0.07) compared to possible-AA-UUC and non-AA-UUC patients. All 14 patients who developed contralateral UUC had aristolactam-DNA adducts; ten of these also had signature mutations. The contralateral UUC-free survival period was shorter in AA-UUC compared to possible- or non-AA-UUC (p=0.019 and 0.002, respectively), whereas no differences among groups were observed for bladder cancer recurrence. In conclusion, AA-UUC patients tend to be younger and female, and have more advanced renal disease. Notably, AA exposure was associated with an increased risk for developing synchronous bilateral and metachronous contralateral UUC., (Copyright © 2013 UICC.)

Predicting patient outcome for colorectal carcinoma (CRC) with lymph node but not distant metastases remains challenging. Various prognostic markers have been identified including microsatellite instability (MSI) and possibly expression of the MHC Class II protein, HLA-DR. About 15% of sporadic CRC exhibits MSI associated with methylation of the DNA mismatch repair gene hMLH1 promoter. In addition, a significant proportion of unselected CRC demonstrates expression of HLA-DR. We sought to examine the relationship between HLA-DR expression, MSI status and prognosis in sporadic Australian Clinicopathological (ACP) Stage C CRC. Two hundred seventy consecutive patients with sporadic ACP Stage C CRC were treated at Concord Repatriation General Hospital between 1986 and 1992. None of these patients received adjuvant chemotherapy and all were followed for a minimum of 5 years or until death. DNA was extracted from paraffin sections and MSI status determined by PCR. HLA-DR expression was determined immunohistochemically using an antibody against the HLA-DR alpha chain. MSI status could be assigned in 235 cases: 176 CRCs (74.9%) were microsatellite stable, whereas 23 (9.8%) had high levels of MSI (MSI-H) and 36 (15.3%) had low levels of MSI (MSI-L). HLA-DR expression by CRC cells was seen in 148 (60.1%) cases and correlated with the presence of tumor-infiltrating lymphocytes (p = 0.0005) and peritumoral lymphocytes (p = 0.003), but not other clinicopathological features or MSI status. HLA-DR-positive CRCs were strongly associated with better patient outcome (p < 0.0001)., (2009 UICC.)

Gene amplification, a common mechanism for oncogene activation in cancers, has been used in the discovery of novel oncogenes. Low-level copy number gains are frequently observed in head and neck squamous cell carcinomas (HNSCCs) where numerous amplification events and potential oncogenes have already been reported. Recently, we applied restriction landmark genome scanning to study gene amplifications in HNSCC and located novel and uncharacterized regions in primary tumor samples. Gain on chromosome 8q22.3, the location of YWHAZ (14-3-3zeta), is found in 30-40% HNSCC cases. Data obtained from fluorescence in situ hybridization and immunohistochemistry on HNSCC tissue microarrays confirmed frequent low-level YWHAZ copy number gain and protein overexpression. YWHAZ mRNA was frequently upregulated in patients' tumor tissues. Furthermore, YWHAZ RNAi significantly suppressed the growth rate of HNSCC cell lines, and overexpression of YWHAZ in HaCaT immortalized human skin keratinocytes promotes overgrowth, as well as morphological changes. Reduced YWHAZ levels increased the G1/G0-phase proportion, decreased the S-phase proportion and the rate of DNA synthesis. Based on this evidence, we suggest that YWHAZ is a candidate proto-oncogene and deserves further investigation into its role in HNSCC carcinogenesis.

High mortality rate for metastatic melanoma is related to its resistant to the current methods of therapy. Melanogenesis is a metabolic pathway characteristic for normal and malignant melanocytes that can affect the behavior of melanoma cells or its surrounding environment. Human melanoma cells in which production of melanin pigment is dependent on tyrosine levels in medium were used for experiments. Peripheral blood mononuclear cells were derived from the buffy coats purchased from Lifeblood Biological Services. Cell pigmentation was evaluated macroscopically, and tyrosinase activity was measured spectrophotometrically. Cell proliferation and viability were measured using lactate dehydrogenase release MTT, [(3)H]-thymidine incorporation and DNA content analyses, and gene expression was measured by real time RT-PCR. Pigmented melanoma cells were significantly less sensitive to cyclophosphamide and to killing action of IL-2-activated peripheral blood lymphocytes. The inhibition of melanogenesis by either blocking tyrosinase catalytic site or chelating copper ions sensitized melanoma cells towards cytotoxic action of cyclophosphamide, and amplified immunotoxic activities of IL-2 activated lymphocytes. Exogenous L-DOPA inhibited lymphocyte proliferation producing the cell cycle arrest in G1/0 and dramatically inhibited the production of IL-1beta, TNF-alpha, IL-6 and IL-10. Thus, the active melanogenesis could not only impair the cytotoxic action of cyclophosphamid but also has potent immunosuppressive properties. This resistance to a chemotherapeutic agent or immunotoxic activity of lymphocytes could be reverted by the action of tyrosinase inhibitors. Thus, the inhibition of melanogenesis might represent a valid therapeutic target for the management of advanced melanotic melanomas.

It is possible that oxidatively damaged DNA which arises as a result of radiotherapy may be involved in the therapeutic effect of the ionizing radiation and in the side effects. Therefore, for the first time, the broad spectrum of oxidatively damaged DNA biomarkers: urinary excretion of 8-oxodG (8-oxo-7,8-dihydro-2'-deoxyguanosine), 8-oxoGua (8-oxo-7,8-dihydroguanine) as well as the level of oxidatively damaged DNA in leukocytes, was analyzed in head and neck cancer patients (n = 27) undergoing fractionated radiotherapy using methodologies which involve HPLC (high-performance liquid chromatography) prepurification followed by gas chromatography with isotope dilution mass spectrometry detection and HPLC/EC. Of all the analyzed parameters in the majority of patients, only urinary excretion of the modified nucleoside significantly increased over the initial level in the samples collected 24 hr after the last fraction. However, for the distinct subpopulation of 10 patients, a significant increase in the level of 8-oxodG in cellular DNA and a simultaneous drop in urinary 8-oxoGua (the repair product of oxidative DNA damage) were detected after completion of the therapy. Because 8-oxoGua is a repair product of the DNA damage, there is a possibility that, at least in the case of some patients with the lowest activity of OGG1 (8-oxo-7,8-dihydroguanine glycosylase), the combination of lower OGG1 repair efficacy and irradiation was associated with increased background level of 8-oxoGua in cellular DNA. Apparently reduced DNA repair is unable to cope with the radiation-induced, and the extra amount of 8-oxoGua leading to an increase of potentially mutagenic/carcinogenic lesions.

The identification of mammary epithelial stem cells raises the hypothesis that these cells may be crucial in the pathogenesis of breast cancer. To further support this, a highly tumourigenic sub-population of cancer cells has recently been identified in primary and metastatic breast cancer samples. In this study, a sub-population of cells displaying features normally attributed to stem cells was identified within the breast cancer cell line MCF-7. This sub-population is capable of growth in anchorage-independent conditions as spherical organoids, displays resistance to proapoptotic agents and significantly greater tumourigenicity than its parental line, with as few as 1,000 cells able to form tumours in immunodeficient mice. Cells within this sub-population can be enriched by serial passages in anchorage-independence, and are characterized by over-expression of the adhesion molecule alpha6-integrin. Alpha-6 integrin proves to be required for the growth and survival of these cells, as the knockdown of ITGA6 causes mammosphere-derived cells to lose their ability to grow as mammospheres and abrogates their tumourigenicity in mice. These findings support the existence of a highly tumourigenic sub-population in breast cancer cells. Furthermore, it shows alpha6-integrin as a potential therapeutic target aimed at tumour-generating subsets of breast cancer cells., (Copyright 2007 Wiley-Liss, Inc.)

Because cost-effectiveness of different cervical cytology screening strategies with and without human papillomavirus (HPV) DNA testing is unclear, we used a Markov model to estimate life expectancy and health care cost per woman during the remaining lifetime for 4 screening strategies: (i) cervical cytology screening at age 32, 35, 38, 41, 44, 47, 50, 55 and 60, (ii) same strategy with addition of testing for HPV DNA persistence at age 32, (iii) screening with combined cytology and testing for HPV DNA persistence at age 32, 41 and 50, iv) no screening. Input data were derived from population-based screening registries, health-service costs and from a population-based HPV screening trial. Impact of parameter uncertainty was addressed using probabilistic multivariate sensitivity analysis. Cytology screening between 32 and 60 years of age in 3-5 year intervals increased life expectancy and life-time costs were reduced from 533 to 248 US Dollars per woman compared to no screening. Addition of HPV DNA testing, at age 32 increased costs from 248 to 284 US Dollars without benefit on life expectancy. Screening with both cytology and HPV DNA testing, at ages 32, 41 and 50 reduced costs from 248 to 210 US Dollars with slightly increased life expectancy. In conclusion, population-based, organized cervical cytology screening between ages 32 to 60 is highly cost-efficient for cervical cancer prevention. If screening intervals are increased to at least 9 years, combined cytology and HPV DNA screening appeared to be still more effective and less costly., (Copyright 2007 Wiley-Liss, Inc.)

Although aneuploidy is commonly observed in human cancers, the molecular mechanism underlying aneuploidization remains unclear. We used multiploid cancer model that had diploid and aneuploid cancer cells within the same cancerous tissue and attempted to detect specific epigenetic alterations associated with tumor cell aneuploidy. Thirty-four multiploid colorectal cancers were subjected to crypt isolation and cell sorting, and paired diploid and aneuploid cancer cells were separated from each cancerous tissue. A methylated CpG island amplification provided a considerable number of CpG sequences that showed different methylation status between the above 2 cell populations. BLAST homology search revealed 24 different candidates (11 hypermethylated and 13 hypomethylated) from these sequences. The putative promoter sequence of the SALL4 (sal-like 4, a human homolog to Drosophila spalt) gene was particularly more frequently hypermethylated in aneuploid cells (62%) than diploid ones (35%) in the 34 multiploid cancers. Moreover, such hypermethylation occurred more often in aneuploid cancers (8 of 16, 50%) than diploid cancers (3 of 18, 17%). In combination with demethylation study on cultured cells, these results implied a possible association between epigenetic silencing of SALL4 and tumor cell aneuploidy. SALL4 may be one of important key players that act as "caretakers" for chromosomal stability. Our new approach is a powerful tool for the global identification of such key players.

Extensive hypermethylation and consecutive transcriptional silencing of tumorsuppressor genes have been documented in multiple tumor entities including breast cancer. In a microarray based genome-wide methylation analysis of five sporadic breast carcinomas we identified a hypermethylated CpG island within the first intron of the prospero related homeobox gene 1 (PROX1). We, therefore, investigated CpG island methylation of PROX1 in a series of 33 pairs of primary breast cancer and corresponding normal tissue samples by bisulfite sequencing and COBRA analyses. Seventeen of these (52%) breast cancer samples revealed a significant accumulation of methylated CpG sites along with a significant reduction of PROX1 transcription compared to normal breast tissues of the same patients. Frequent methylation was also observed in brain metastases from primary breast cancer (21/37 = 57% of cases). Secondary, we analysed 38 brain metastases of primary breast carcinomas and detected a significantly reduced expression of PROX1 compared to normal breast tissue (p < 0.001) and primary breast carcinomas (p < 0.05), respectively. Additionally, treatment of breast cancer cell lines with demethylating agents could reactivate PROX1 transcription. In summary, we have identified PROX1 as a novel target gene that is hypermethylated and transcriptionally silenced in primary and metastatic breast cancer., (Copyright (c) 2007 Wiley-Liss, Inc.)

Previous studies have indicated that lymphangiogenesis in solid tumors is associated with lymphatic metastasis. Overexpression of Vascular endothelial growth factor (VEGF)-C plays a major role in lymphangiogenesis in cancers. In the present study, DNA methylation and expression of the VEGF-C gene was investigated in gastric cancer (GC). Four GC cell lines (MKN-45, MKN-74, HSC-39 and HSC-43) showed no expression of VEGF-C, and the VEGF-C gene was found to be methylated in these cells. In contrast, 7 GC cell lines (MKN-1, MKN-7, MKN-28, TMK-1, KATO-III, SH101-P4 and HSC-44PE) expressed VEGF-C, and the VEGF-C gene was found to be unmethylated in these cell lines. In addition, expression of VEGF-C mRNA was retrieved by treatment with a demethylating agent, Aza-2'-deoxycytidine. In GC tissue samples, bisulfite DNA sequencing analysis revealed that VEGF-C was not methylated in 9 (29.0%) of 31 GC samples, whereas demethylation was not observed in corresponding non-neoplastic mucosa samples. Overexpression of VEGF-C mRNA was observed in 16 (51.6%) of 31 GC samples by quantitative reverse transcription-polymerase chain reaction. Of the 9 GC cases with VEGF-C demethylation, 8 (88.9%) overexpressed VEGF-C. In contrast, of the 22 GC cases without VEGF-C demethylation, 8 (36.4%) overexpressed VEGF-C (p = 0.0155). Furthermore, lymphatic vessel density determined by immunostaining of podoplanin in GC tissues was associated with overexpression of VEGF-C (p < 0.0001). These results suggest that demethylation and activation of the VEGF-C gene is likely involved in lymphangiogenesis in GC., ((c) 2007 Wiley-Liss, Inc.)

We recently reported the potential of genistein in augmenting gemcitabine-induced killing of pancreatic cancer (Banerjee, S. et al., Cancer Research 2005;65:9064-72). Since cis-diaminedichloroplatinum (II) (cisplatin) is widely used against solid tumors, we further investigated whether genistein pretreatment could be used as a novel strategy for cisplatin-induced killing of pancreatic cancer cells in vitro and enhanced antitumor activity in vivo. Our in vitro results showed that pretreatment of cells with genistein followed by cisplatin resulted in significant loss of cell viability and potentiated apoptosis irrespective of the metastatic ability of cells. Mechanistically, genistein augmented cisplatin induced killing by down regulating transcription factor-NF-kappaB and anti-apoptotic Akt expression. NF-kappaB was found upregulated when pancreatic cancer cells were exposed to cisplatin, suggesting the potential mechanism of acquired chemo-resistance. In addition, we also showed, for the first time, that genistein in combination with cisplatin is more effective antitumor agent in our orthotopic tumor model. But most importantly, our data also showed that a specific target, such as NF-kappaB, was inactivated in animal tumors treated with genistein and cisplatin. Immunohistochemical data showed reduced staining for phospho-p65, Bcl-xL and MMP-9 in treated tumors compared to control tumors, but the lowest activity was seen in the combination group. These results provide strong molecular in vivo evidence in support of our hypothesis that inactivation of the NF-kappaB signaling pathway by genistein results in the chemo-sensitization of pancreatic tumors to cisplatin, which is likely to be an important and novel strategy for the treatment of pancreatic cancer.

Insulin-like growth factor binding protein-3 (IGFBP-3) is postulated to be a mediator of growth suppression signals. Here, we examined the methylation status of IGFBP-3 to correlate to clinicopathological factors in human cancers. The methylation status of IGFBP-3 was determined by bisulfite DNA sequencing and was correlated with expression semi-quantified by real-time RT-PCR to develop a methylation-specific PCR (MSP) assay for IGFBP-3. Using the MSP assay, we examined the methylation status of IGFBP-3 in gastric cancer (GC), colorectal cancer (CRC), breast cancer (BC) and malignant mesothelioma (MM). IGFBP-3 methylation was detected in 6 of 13 (46%) and 16 of 24 (67%) GC cell lines and tumors, respectively; 4 of 8 (50%) and 15 of 26 (58%) CRC cell lines and tumors, respectively; 3 of 11 (27%) and 7 of 39 (18%) BC cell lines and tumors, respectively and 1 of 5 (20%) and 18 of 56 (32%) MM cell lines and tumors, respectively. Interestingly, the methylation status of MM specimens from Japanese patients (75%, 12 out of 16 patients) was significantly higher than those from the USA (15%, 6 out of 40 patients) (p < 0.0001), suggesting the presence of ethnic differences in the IGFBP-3 methylation status. We also found that IGFBP-3 methylation was preferentially present in GCs arising in the lower-third of the stomach (p = 0.079). In summary, our results showed that IGFBP-3 methylation played an important role in the silencing of its expression, suggesting that IGFBP-3 may act as a tumor suppressor gene in several human cancers examined.

RASSF2 can bind directly to K-Ras and function as a negative effector of Ras protein. RASSF2A is the only isoform of RASSF2 that contains CpG islands in its promoter and it has been reported to be inactivated by its promoter methylation in several human cancers. In the present study, we investigated the correlation of RASSF2A expression with its promoter methylation in nasopharyngeal carcinoma (NPC). Expression of RASSF2A was down-regulated in 80% (4/5) of NPC cell lines. Decreased RASSF2A expression was also observed in NPC primary tumors compared with normal nasopharyngeal epithelia. Promoter methylation of RASSF2A could be detected in all the RASSF2A-silenced cell lines (4/5) of the NPC cell lines and 50.9% (27/53) of primary tumors, but not in any of the normal epithelia. RASSF2A-methylated cases showed a significantly lower level of RASSF2A expression than unmethylated cases. Loss of RASSF2A expression can be greatly restored by the methyltransferase inhibitor 5-aza-dC in NPC cell lines. In addition, patients with methylated RASSF2A presented a higher frequency of lymph node metastasis (p < 0.05). Ectopic expression of RASSF2A in RASSF2A-silenced and -methylated NPC cell line CNE2 shows that RASSF2A could inhibit cell cycle progression, colony formation and cell migration, which provided further evidence that RASSF2A is a candidate tumor suppressor gene. In conclusion, RASSF2A, a candidate tumor suppressor gene (TSG), is frequently inactivated by its promoter methylation and this aberrant methylation correlates with lymph node metastasis in NPC.

Epigenetic silencing of functionally important genes is important in the development of malignancies and is a source of potential markers for molecular detection. Primary central nervous system lymphoma (PCNSL) is an increasingly common tumor that has not been extensively examined for changes in promoter region methylation. We examined 14 tumor suppressor genes in 25 cases of PCNSL using methylation-specific PCR. Methylation was observed in DAPK (84%), TSP1 (68%), CRBP1 (67%), p16(INK) (4a) (64%), p14(ARF) (59%), MGMT (52%), RARbeta2 (50%), TIMP3 (44%), TIMP2 (42%), p15(INK) (4b) (40%), p73 (28%), hMLH1 (12%), RB1 (8%) and GSTP1 (8%). Promoter methylation of p14(ARF), p16(INK) (4a) and MGMT was correlated with loss of expression by immunohistochemical staining. The methylation of many of these genes in PCNSL is similar to that reported in other high-grade B-cell lymphomas. All 25 cases of PCNSL had methylation of at least 2 genes. Methylation of DAPK, p16(INK) (4a) or MGMT was found in 96% of the tumors, suggesting simple marker strategies to detect circulating methylated DNA in serum that might facilitate early tumor detection. Our study provides insight into the epigenetic alterations in PCNSL and provides potential biomarkers of disease.

The role of aberrant tumor suppressor gene methylation in the aggressiveness of papillary thyroid cancer (PTC) has not been documented. By showing promoter methylation-induced gene silencing in PTC-derived cell lines, we first demonstrated the functional consequence of methylation of several recently identified tumor suppressor genes, including those for tissue inhibitor of metalloproteinase-3 (TIMP3), SLC5A8, death-associated protein kinase (DAPK) and retinoic acid receptor beta2 (RARbeta2). We then investigated the role of methylation of these genes in the aggressiveness of PTC by examining the relationship of their aberrant methylation to clinicopathological characteristics and BRAF mutation in 231 primary PTC tumors. Methylation of TIMP3, SLC5A8 and DAPK was significantly associated with several aggressive features of PTC, including extrathyroidal invasion, lymph node metastasis, multifocality and advanced tumor stages. Methylation of these genes was also significantly associated with BRAF mutation in PTC, either individually or collectively in various combinations. Methylation of these genes, either individually or collectively, occurred more frequently in more aggressive classical and tall-cell PTC subtypes than in less aggressive follicular-variant PTC, with the latter known to infrequently harbor BRAF mutation. Several other tumor suppressor genes investigated were not methylated. These results suggest that aberrant methylation and hence silencing of TIMP3, SLC5A8, DAPK and RARbeta2, in association with BRAF mutation, may be an important step in PTC tumorigenesis and progression.

Survivin, a member of the inhibitor of apoptosis protein family, is expressed in most human cancers, but undetectable in normal differentiated adult tissue in vivo. Because of this cancer-related expression, survivin is a promising target for cancer therapy. To determine the expression and prognostic role of survivin in acute myeloid leukemia (AML), we investigated the mRNA expression pattern of survivin and of the splice variants survivin-2B and survivin-DeltaEx3 in adult (n = 74) and children (n = 31) with de novo AML using RT-PCR. Survivin was the predominant transcript variant in AML cells, whereas significantly lower levels of survivin-2B and survivin-DeltaEx3 were observed (p < or = 0.0001). Neither expression of survivin nor of any splice variant correlated with maturation stage (FAB subtypes, immunophenotype) or cytogenetic risk groups. For AML cases treated according to AMLCG92 (adult) and AML-BFM93 (children) protocols, respectively, expression patterns were correlated with clinical data: in adult AML (n = 51), low expression of survivin-2B correlated with a better overall survival (p = 0.05; mean survival time 19 months vs. 9 months) and a better eventfree survival (p < or = 0.01; 27 months vs. 10 months). In childhood AML (n = 31), high survivin-DeltaEx3 expression was associated with a shorter overall survival (p < or = 0.05; 24 months vs. 43 months). We conclude that certain survivin splice variants have potential prognostic impact for long-term therapy outcome in adult as well as childhood de novo AML.

Promotor hypermethylation is a common event in human cancer. O6-Methylguanine-DNA Methyltransferase (MGMT) is a gene involved in DNA repair, which is methylated in a variety of cancer types. In colorectal cancer and lung cancer, hypermethylation of MGMT has been correlated with p53 mutation. In the present study, 132 samples of esophageal adenocarcinoma and 58 samples of normal esophageal tissue were investigated for MGMT hypermethylation status by methylation-specific real-time PCR and results were correlated to clinicopathological parameters, patient's survival, p53 mutation and expression of p53 protein and MGMT protein. In the carcinomas, hypermethylation of MGMT was found in 63.6% of cases and loss of MGMT protein expression in 48.5% of cases. Furthermore, MGMT hypermethylation was found in 5.7% of normal esophageal smooth muscle tissue, in 20.0% of esophageal squamous epithelium and in 61.5% of nonneoplastic Barrett's mucosa. In the carcinomas, hypermethylation of the MGMT gene was correlated with loss MGMT protein expression (p < 0.0001) and with high tumor differentiation (p = 0.0079). In contrast, no correlation between MGMT hypermethylation, Lauren's classification, WHO classification, tumor size, gender, age, pT category and pN category, and p53 status was found. Neither MGMT hypermethylation nor loss of MGMT protein expression was correlated with patient's survival. In conclusion, MGMT hypermethylation in esophageal adenocarcinoma is a frequent event that is associated with loss of MGMT protein expression but not with patient's outcome., (2006 Wiley-Liss, Inc.)

Folate receptor alpha (FRalpha) is a membrane-bound protein with high affinity for binding and transporting physiologic levels of folate into cells. Folate is a basic component of cell metabolism and DNA synthesis and repair, and rapidly dividing cancer cells have an increased requirement for folate to maintain DNA synthesis, an observation supported by the widespread use of antifolates in cancer chemotherapy. FRalpha levels are high in specific malignant tumors of epithelial origin compared to normal cells, and are positively associated with tumor stage and grade, raising questions of its role in tumor etiology and progression. It has been suggested that FRalpha might confer a growth advantage to the tumor by modulating folate uptake from serum or by generating regulatory signals. Indeed, cell culture studies show that expression of the FRalpha gene, FOLR1, is regulated by extracellular folate depletion, increased homocysteine accumulation, steroid hormone concentrations, interaction with specific transcription factors and cytosolic proteins, and possibly genetic mutations. Whether FRalpha in tumors decreases in vivo among individuals who are folate sufficient, or whether the tumor's machinery sustains FRalpha levels to meet the increased folate demands of the tumor, has not been studied. Consequently, the significance of carrying a FRalpha-positive tumor in the era of folic acid fortification and widespread vitamin supplement use in countries such as Canada and the United States is unknown. Epidemiologic and clinical studies using human tumor specimens are lacking and increasingly needed to understand the role of environmental and genetic influences on FOLR1 expression in tumor etiology and progression. This review summarizes the literature on the complex nature of FOLR1 gene regulation and expression, and suggests future research directions., (2006 Wiley-Liss, Inc.)

Regional DNA hypermethylation and global DNA hypomethylation are 2 epigenetic alterations associated with colorectal cancers. However, their correlation with microsatellite instability (MSI) and chromosomal instability (CIN) in colorectal cancer, and their relationship with chromatin conformation and histone modification are not clear. In this study, we analyzed regional and global methylation in 16 cell lines and 64 primary colorectal cancers. We found that MSI and CIN are 2 alternative events in most cell lines and tumors. Furthermore, regional hypermethylation and global hypomethylation are also alternative events in most cases. We also observed a strong correlation between MSI and regional hypermethylation and between CIN and global hypomethylation. We further analyzed chromatin conformation and histone acetylation in cell lines with CIN or MSI. CIN cancers had open chromatin conformation and enriched histone acetylation in repetitive as well as in gene-specific regions. MSI cancers, on the other hand, had closed chromatin conformation and low levels of histone acetylation. After a MSI cell line was treated with 5-aza-2'-deoxycytidine or trichostatin A, the closed chromatin conformation became open, and histone acetylation was enriched. These observations support our hypothesis that in colorectal cancer, regional hypermethylation and global hypomethylation are associated with altered chromatin conformation and histone acetylation, which might have a causal correlation with MSI and CIN, respectively., (Copyright 2006 Wiley-Liss, Inc.)

We evaluated the aberrant promoter methylation profile of a panel of 3 genes in DNA from tumor and sputum samples, in view of a complementary approach to spiral computed tomography (CT) for early diagnosis of lung cancer. The aberrant promoter methylation of RARbeta2, p16(INK4A) and RASSF1A genes was evaluated by methylation-specific PCR in tumor samples of 29 CT-detected lung cancer patients, of which 18 had tumor-sputum pairs available for the analysis, and in the sputum samples from 112 cancer-free heavy smokers enrolled in a spiral CT trial. In tumor samples from 29 spiral CT-detected patients, promoter hypermethylation was identified in 19/29 (65.5%) cases for RARbeta2, 12/29 (41.4%) for p16(INK4A) and 15/29 (51.7%) for RASSF1A. Twenty-three of twenty-nine (79.3%) samples of the tumors exhibited methylation in at least 1 gene. In the sputum samples of 18 patients, methylation was detected in 8/18 (44.4%) for RARbeta2 and 1/18 (5%) for both RASSF1A and p16(INK4A). At least 1 gene was methylated in 9/18 (50%) sputum samples. Promoter hypermethylation in sputum from 112 cancer-free smokers was observed in 58/112 (51.7%) for RARbeta2 and 20/112 (17.8%) for p16, whereas methylation of the RASSF1A gene was found in only 1/112 (0.9%) sputum sample. Our study indicates that a high frequency of hypermethylation for RARbeta2, p16(INK4A) and RASSF1A promoters is present in spiral CT-detected tumors, whereas promoter hypermethylation of this panel of genes in uninduced sputum has a limited diagnostic value in early lung cancer detection., ((c) 2005 Wiley-Liss, Inc.)

A G to T mutation at nucleotide position 2128 in the human FGFR3b coding region resulting in a Cys for Gly substitution (G697C) in the tyrosine kinase domain was observed in 62% (44/71) of oral squamous cell carcinomas (OSCC) examined. Immunostained FGFR3b was found in the cytoplasm of prickle cells in normal epithelia, and FGFR3b was localized in the cytoplasm and nucleus in non-FGFR3b mutant OSCC. Overexpressed FGFR3b protein on plasma membranes was noted in OSCC bearing the FGFR3b mutation. Enhanced tyrosine kinase activity of G697CFGFR3b was confirmed. Our results indicate that G697C is an activating mutation causing constitutive ligand-independent FGFR3b signaling. This mutation may be involved in the progression of OSCC and thus the FGFR3b coding sequence may have diagnostic or prognostic value for OSCC., (Copyright (c) 2005 Wiley-Liss, Inc.)

Reprimo is a new candidate mediator of p53-mediated cell cycle arrest at the G2 phase. Loss of Reprimo gene expression accompanied by its promoter methylation was identified in pancreatic and lung cancers. Our aim was to examine the methylation status of Reprimo in a broad range of cancers. We examined Reprimo expression by RT-PCR and the DNA methylation status of the Reprimo promoter by MSP in 39 tumor cell lines. Loss or downregulation of Reprimo expression was frequent (62%), and we confirmed that transcriptional repression of Reprimo was caused by hypermethylation (overall concordance 92%). Treatment of expression-negative cells with 5-aza-2'-deoxycytidine restored Reprimo expression. We then examined aberrant methylation of Reprimo in 645 tumors representing 16 tumor types. Promoter methylation of Reprimo was found in 79% of gastric cancers, 62% of gallbladder cancers, 57% of lymphomas, 56% of colorectal cancers, 40% of esophageal adenocarcinomas, 37% of breast cancers and 31% of leukemias. Methylation frequencies in ovarian cancers, bladder cancers, cervical cancers, brain tumors, malignant mesotheliomas and pediatric tumors were lower (0-20%). Reprimo methylation was rarely detected in nonmalignant tissues (0-11%) except for gastric epithelia. While colorectal polyps were also frequently methylated (27%), chronic cholecystitis samples were infrequently methylated (4%). Furthermore, we failed to identify Reprimo mutation in colorectal and gastric cancer cell lines and 50 primary colorectal cancers. Aberrant methylation of Reprimo with loss of expression is a common event and may contribute to the pathogenesis of some types of human malignancy., (Copyright 2005 Wiley-Liss, Inc.)

Beyond human papillomavirus (HPV) infection, host genetic factors may contribute to cervical carcinogenesis. This study aims to test the hypothesis that CA-dinucleotide repeat polymorphism in the first intron of the interferon-gamma (IFN-gamma) gene is associated with HPV-initiated cervical carcinogenesis. A hospital-based case-control study including patients with low-grade squamous intraepithelial lesions (LSILs; n = 93), high-grade squamous intraepithelial lesions (HSILs; n = 123) and invasive carcinomas (n = 153) of the uterine cervix, as well as 1:1 age-matched controls, was conducted. The IFN-gamma genotype was determined by PCR and capillary electrophoresis with internal standards. HPV genotype was determined by consensus PCR and reverse line blot hybridization. Genotypes containing the 12 or 14 allele (12 or 14 CA repeats) were significantly more common in patients with HSILs than in controls (46% vs. 22%; OR = 3.0; 95% CI = 1.7-5.2; p < 0.0001). In contrast, genotypes containing 13 and 18 were significantly more common in controls than in patients with HSILs (76% vs. 53%; OR = 0.3; 95% CI = 0.2-0.6; p = 0.0001) or squamous cell carcinomas (74% vs. 63%; OR = 0.6; 95% CI = 0.4-1.0; p = 0.037). The frequency of the 12 and 14 genotypes increased significantly in accordance with the severity of cervical carcinogenesis (p(test for trend) = 0.0002), whereas the 13 and 18 genotypes showed the opposite trend (p(test for trend) = 0.007). Comparing IFN-gamma genotype and HPV status, 18-containing genotypes were more frequently found in HPV(+) LSILs, and 12-containing genotypes were less frequently found in HPV(+) HSILs. Compared with non-13 genotypes, 13 genotype HSILs were more frequently infected with HPV58 (70% vs. 45%) and less frequently infected with HPV18 (0% vs. 16%; p= 0.007). Genetic polymorphism of the IFN-gamma gene is associated with individual susceptibility to cervical carcinogenesis. This polymorphism correlates with HPV infection in a disease- and type-specific manner., ((c) 2004 Wiley-Liss, Inc.)

The human DOC-2/DAB2 interactive protein gene (hDAB2IP) is a novel member of the Ras GTPase-activating gene family that is known to act as a tumor suppressor gene and is inactivated by methylation in prostate and breast cancers. We established previously a methylation-specific PCR (MSP) for the promoter region (m2a and m2b regions) of hDAB2IP and examined hDAB2IP methylation status in breast cancers. We analyzed the methylation and expression status of hDAB2IP in lung cancers. The methylation status of hDAB2IP was examined in lung cancer cell lines using bisulfite sequencing and MSP. Expression was examined using conventional and real-time RT-PCR, and methylation was found to be inversely correlated with expression, confirming that the MSP can also be used to examine hDAB2IP methylation status in lung cancers. Aberrant methylation was detected at the m2a region in 19 of 47 lung cancer cell lines (40%) and 26 of 70 primary tumors (37%) and at the m2b in 16 lines (34%) and 25 of 70 tumors (36%). Gene expression was restored in methylated cell lines supplemented with 5-aza-2'-deoxycytidine, confirming that methylation was responsible for downregulation. We also examined the relationship between hDAB2IP methylation and clinico-pathological features of the lung cancers and found that hDAB2IP methylation was associated with advanced disease stage. Our results demonstrate that hDAB2IP methylation is frequently present in lung cancers and plays a key role in hDAB2IP silencing. hDAB2IP methylation could be used as a biomarker for disease stage, reflecting the degree of clinico-pathological malignancy of lung cancer.

Cell-Cell communication via gap junctions plays a key role in carcinogenesis and in growth control. One of the gap junction proteins, Connexin 26 (Cx26) was considered as tumor suppressor in various cancers. In our study, the expression of Cx26 was analyzed in human lung cancer. The reduced mRNA expression was observed in 17 lung cancer cell lines examined by Northern blot analysis and RT-PCR. In 138 primary carcinomas comprising all subtypes analyzed by immunohistochemistry, 85 cases (62%) exhibited no expression of Cx26, whereas in other 53 cases the Cx26 staining was positive (38%). Additionally, an association between Cx26 protein expression and higher grading of tumors was found in whole tumor samples (p =0.028) but no statistically significant correlations could be observed with tumor stage, tumor size and node status. In squamous cell carcinoma, tumors with higher stage and grading were linked to higher expression of Cx26 (p = 0.015 and 0.017, respectively). To explore the mechanism responsible for the downregulation of Cx26, we treated 2 lung cancer cell lines H2170 and H226 with the demethylation agent 5-aza-2'-deoxycytidine and found the reexpression of Cx26 mRNA. Methylation status of these 2 cell lines was further analyzed by PCR amplification of bisulfite modified DNA and sequencing. A heterogeneous methylation pattern turned out. Our results suggest the inactivation of Cx26 in lung cancer may be explained by promoter methylation.

Suppressor of cytokine signaling (SOCS)-1 inhibits signaling of the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway by several cytokines and has tumor suppressor activity. Methylation of the SOCS-1 CpG island has been shown to inactivate the SOCS-1 gene in certain human cancers. In our study, we investigated methylation status of the SOCS-1 gene by methylation-specific PCR in 75 gastric carcinoma (GC) tissues, 25 corresponding nonneoplastic mucosae and 10 normal gastric mucosae from healthy young individuals. We also performed bisulfite sequencing of DNAs from 2 GC tissues. In addition, SOCS-1 mRNA levels were examined in 50 GCs by quantitative RT-PCR. Hypermethylation of the SOCS-1 gene was detected in 33 (44%) of 75 GC tissues and in 3 (12%) of 25 corresponding nonneoplastic mucosae; the incidence was significantly different (p = 0.004). None of the 10 normal gastric tissues from healthy individuals showed hypermethylation. Methylation of the SOCS-1 gene was associated with lymph node metastasis, advanced tumor stage and reduced expression of SOCS-1 in GC tissues (p = 0.009, 0.034 and 0.002, respectively). Reduced expression of SOCS-1 in GC tissues was associated with lymph node metastasis and advanced tumor stage (p = 0.013 and 0.002, respectively). Our results suggest that transcriptional inactivation of the SOCS-1 gene by hypermethylation may be involved in development, progression and metastasis of GC., ((c) 2004 Wiley-Liss, Inc.)

We have previously mapped a putative prostate cancer tumor-suppressor gene to a 1-2 Mb region of 12p12-13. Initial work to identify the tumor suppressor at this locus focused on candidates previously implicated in malignancy; however, mutational and methylation analyses failed to identify significant genomic events. An alternative approach is to use expression analysis to prioritize the genes within the region of interest. This experimental design is based on the hypothesis that tumor-suppressor genes demonstrate decreased expression in tumors compared to normals. Herein, we narrow the region of interest using deletion mapping data and employ expression analysis to prioritize the genes in the minimal deleted region. Highly informative polymorphic markers spanning our region were used to assess for loss of heterozygosity in 99 tumor and normal DNA pairs. The minimal region of deletion was determined to be approximately 500 kb bounded by D12S391 and A002Q26. Publically available databases place 7 genes within this minimal deletion region. An additional 3 genes lie just outside this minimal deletion region and could possibly be inactivated by deletion of promoter, 3'-untranslated region sequences or alternative splice variants. Relative levels of expression of these 10 candidate genes were determined in 6 normal prostates, 5 local prostate tumors, 9 prostate lymph node metastases, 6 prostate cancer cell lines and 12 prostate cancer xenografts using quantitative RT-PCR. DUSP16, FLJ10298 and BCLG were significantly downregulated in both clinical tumors and cultured prostate cancer tissue, indicating that one or all may be critical to initiation or progression of prostate carcinoma., (Copyright 2004 Wiley-Liss, Inc.)

We have identified 2 antigens recognized by several melanoma-specific cytolytic T lymphocyte clones isolated from a melanoma patient with a clinical history of tumor regression after immunotherapy. Both antigens are presented by HLA-A2 and encoded by gene MAGE-C2, a cancer-germline gene shown previously to be silent in normal somatic tissues and expressed in 40% of melanomas and in other tumor types. One antigen corresponds to peptide ALKDVEERV(336-344), whereas the other corresponds to peptide LLFGLALIEV(191-200). The CTL clones recognizing these 2 peptides also recognized allogeneic tumor cell lines expressing MAGE-C2 and HLA-A2. These 2 new peptides are the first known MAGE-C antigens and represent promising targets for cancer immunotherapy., (Copyright 2004 Wiley-Liss, Inc.)

Neuroblastoma is the most common pediatric solid tumor. Although many allelic imbalances have been described, a bona fide tumor suppressor gene for this disease has not been found yet. In our study, we analyzed 2 genes, PTEN and DMBT1, mapping 10q23.31 and 10q25.3-26.1, respectively, which have been found frequently altered in other kinds of neoplasms. We screened both genes for homozygous deletions in 45 primary neuroblastic tumors and 12 neuroblastoma cell lines. Expression of these genes in cell lines was assessed by RT-PCR analysis. We could detect 2 of 41 (5%) primary tumors harboring PTEN homozygous deletions. Three of 41 (7%) primary tumors and 2 of 12 cell lines presented homozygous losses at the g14 STS on the DMBT1 locus. All cell lines analyzed expressed PTEN, but lack of DMBT1 mRNA expression was detected in 2 of them. We tried to see whether epigenetic mechanisms, such as aberrant promoter hypermethylation, had any role in DMBT1 silencing. The 2 cell lines lacking DMBT1 expression were treated with 5-aza-2'-deoxycytidine; DMBT1 expression was restored in only one of them (MC-IXC). From our work, we can conclude that PTEN and DMBT1 seem to contribute to the development of a small fraction of neuroblastomas, and that promoter hypermethylation might have a role in DMBT1 gene silencing., (Copyright 2004 Wiley-Liss, Inc.)

Palladacycle compounds obtained from N, N-dimethyl-1-phenethylamine (dmpa), phenyl-2-pyridinyl-acetylene and 1-phenyl-3-N, N-dimethylamine-propine, respectively, were complexed to 1, 2 ethanebis (diphenylphosphine) (dppe) ligand to synthesize antitumor cyclopalladated complexes that were tested in vitro and in vivo against syngeneic B16F10-Nex2 murine melanoma cells of low immunogenicity implanted subcutaneously in mice. Complexes were not toxic to mice injected 3 times i.p. with as much as 60 microM/animal/week. Of 3 cyclopalladated complexes that were inhibitory in vitro at low concentrations (<1.25 microM), complex 7a was the most active in vivo, delaying tumor growth and prolonging animal survival. In vitro, binucleate complex 7a caused a collapse of respiratory activity with an abrupt decrease of extracellular acidification on short incubation (up to 100 min), followed by DNA degradation after 24 hr. The apoptosis-like reaction to this Pd-complex was not accompanied by increased levels of caspases 1 and 3. Complex 7a bound to a bacterial plasmid DNA, causing late conformational changes after 24 hr. Two other complexes with different C, N-cycles were also apoptotic and 2 binucleated ones were inactive. These results introduce the palladacycle-dppe complexes as promising antitumor drugs with exquisite structural specificities and for action in vivo and in vitro., (Copyright 2003 Wiley-Liss, Inc.)

Previously, we have identified aloe-emodin (AE) as a new type of anticancer agent, with activity that is based on apoptotic cell death promoted by a neuroectodermal tumor-specific drug uptake. We attempt to clarify the intracellular target of AE and the apoptosis-signaling pathway activated by AE in neuroblastoma cell lines. Two-photon excitation microscopy and spectroscopic titrations documented that AE is highly concentrated in susceptible cells and binds to DNA. One of the most important mediators of apoptotic response to genotoxic stimuli, such as anticancer agents, is the p53 tumor suppressor gene. To evaluate the role played by p53 in AE-induced apoptosis a p53 mutant cell line, which lacks transcriptional activity of p53 targeted genes, was tested. AE displayed a reduced growth inhibitory and pro-apoptotic activity in p53 mutant cells (SK-N-BE(2c)) with respect to the p53 wild-type line (SJ-N-KP). This effect was not caused by a reduced drug uptake in the mutant neuroblastoma cell line but was related to a different apoptotic cell phenotype. Whereas SJ-N-KP cells were susceptible to a p53 transcription-dependent pathway of apoptosis, SK-N-BE(2c) cells underwent apoptosis with up-regulation of p53 expression but not of p53-target genes. After AE treatment p53 translocates to the mitochondria inter-membrane space in both neuroblastoma cell lines. Due to its high accumulation in neuroectodermal tumor cells AE could also kill tumor cells harboring p53 mutant genes. This property would further contribute to AE specific anti-tumor activity and might be exploitable in the clinic., (Copyright 2003 Wiley-Liss, Inc.)

Hypermethylation of cytosines in CpG-rich islands of the promoter regions of regulatory genes has been discovered as a common mechanism of gene silencing during carcinogenesis. We analysed 64 primary lung carcinomas for promoter methylation of the tumour suppressor genes (TSGs) p16 (p16(INK4a)/CDKN2A) and p14 (p14(ARF)) by methylation-specific PCR, in order to evaluate aberrant methylation as a potential biomarker for epigenetic alterations in tobacco-related lung cancer. Methylation of p16 was observed in 34% (22/64) of the lung tumours examined. In particular, p16 methylation occurred in nonsmall cell lung cancer (NSCLC) only, with 41 % (22/54) of the tumours being positive. The highest frequency was found in large cell carcinoma (5/7, 71%), followed by adenocarcinoma (9/25, 36%) and squamous cell carcinoma (7/21, 33%). Methylation of the p14 gene was less frequent in lung cancer (4/52, 8%). When association with tobacco smoking was analysed, 42% (21/50) of NSCLC from ever smokers exhibited p16 methylation. Interestingly, the analysis revealed a significantly higher risk of p16 methylation in former smokers as compared to current smokers [odds ratio (OR) 5.1; 95% confidence interval (CI) 1.3-22]. The difference was retained after adjustment for age (OR 3.7; 95% CI 0.9-17). The promoter methylation results were then combined with data on genetic alterations determined previously in the same set of tumours. This data similarly showed that p16 methylation in parallel with p53 gene mutation or p14 methylation occurred more frequently in former smokers than in current smokers (44% vs. 14%; P = 0.035). Taken together, our data suggest that analysis of promoter methylation in TSGs may provide a valuable biomarker for identification of groups with an elevated risk of cancer, such as smokers and ex-smokers., (Copyright 2003 Wiley-Liss, Inc.)

Capecitabine is an orally available fluoropyrimidine and is finally converted to 5-FU selectively in tumor tissues. In our study, we examined whether the antitumor activity of capecitabine is directly affected by a modulation of dihydropyrimidine dehydrogenase (DPD). The modulations were carried out by the overexpression of DPD in tumor cells and by tumor selective DPD inhibition. The DPD-overexpressing cells were obtained by transfection of human DPD cDNA into HCT116 human colorectal cancer cells. The HCT116 cells bearing DPD cDNA expressed about 13 times higher DPD activities than the parental HCT116 cells, and they became significantly less susceptible to capecitabine than the parental cells when transplanted into nude mice. Administration of RO0094889 that is converted to a DPD inhibitor 5-vinyluracil selectively in tumor tissues restored the antitumor activity of capecitabine against the tumor of the HCT116 cells carrying DPD cDNA and various tumors expressing DPD. As compared to 5-ethynyluracil or 5-vinyluracil, which inhibited DPD not only in tumor tissues but also in other non-cancerous tissues, the effective dose range of RO0094889 in augmenting the efficacy of capecitabine was much broader. These results indicate that the antitumor activity of capecitabine is directly affected by DPD activities in tumor tissues and therefore, the combination of capecitabine and a tumor selective DPD inhibitor, RO0094889, will be beneficial to patients who have tumors with high levels of DPD., (Copyright 2003 Wiley-Liss, Inc.)

In this report the proportion of consecutive and familial breast cancer cases harboring the 657del5 of exon 6 of the NBS1 gene was determined to assess whether it is associated with the increased risk of breast cancer development. The study consisted of 3 groups of patients: a series of consecutive 150 patients with histologically confirmed breast cancer, diagnosed under the age of 50 in the city of Szczecin; a series of 80 breast cancer patients with a family history of breast cancer in their first-degree relatives; and a series of 530 consecutive individuals without the diagnosis of breast cancer selected at random by family doctors from the city of Szczecin. Molecular examination included allele-specific PCR assay for the common Slavic NBS1 mutation (657del5), LOH analysis using denucleotide CA repeat microsatellite markers, haplotype analysis and sequencing. The NBS1 founder mutation was detected in 2 of 150 (1.3%) consecutive breast cancer cases diagnosed under the age of 50 years; in 3 of 80 familial breast cancer cases (3.7%); and in 3 of 530 individuals (0.6%) from the general population. Examination of tumor DNA from patients with the NBS1 mutation (groups A and B) revealed loss of heterozygosity (LOH) in all cases. Additional haplotype analysis revealed that allelic loss affects specifically wild-type alleles. The majority of probands with breast cancer and the NBS1 mutation had a positive family history of breast cancer in their first-degree relatives. It appears that the 657del5 mutation in exon 6 of the NBS1 gene is responsible for the occurrence of a small but significant proportion of familial breast cancer patients., (Copyright 2003 Wiley-Liss, Inc.)

Loss of heterozygosity (LOH) at chromosome 3p21 is frequent in cervical cancers. The candidate tumor suppressor gene, RASSF1A located at 3p21.3, is found to be inactivated in several major human cancers, implicating its significance in carcinogenesis. We aimed to investigate the status of RASSF1A in cervical cancers. The mutation and methylation status of RASSF1A were analysed in 4 cervical cancer cell lines, 50 primary cervical cancers including 33 squamous cell carcinoma (SCC), 17 adenocarcinoma (AC) and 11 normal controls. The primary cancer samples were also detected for LOH at 3p21 and human papillomavirus (HPV). Hypermethylation of RASSF1A was detected in 30% of SCC, 12% of AC and in 1 of the 4 cancer cell lines but was absent in all normal cases. Methylation of the cancer cell line was associated with loss of gene expression, which was restored by demethylation. About 67% (8 of 12) of hypermethylated primary cancers showed concomitant LOH at 3p21. No somatic mutation was found in all primary cancer samples or cell lines but 2 cases showed germline polymorphism at codon 133. Oncogenic HPV DNAs were found in most cancer samples. No correlation was detected between RASSF1A-hypermethylation or LOH at 3p21 and age of patient, HPV genotype, tumor grade and stage. Hypermethylation of RASSF1A occurs in a subset of cervical cancers, among which concomitant LOH at 3p21 is common. The results supported that RASSF1A may be one of the cervical cancer-related tumor suppressor genes located at 3p21 regions., (Copyright 2003 Wiley-Liss, Inc.)

We previously reported on the amplification and overexpression of the mouse double minute 4 homolog gene (MDM4) from 1q32 in a subset of malignant gliomas (Riemenschneider et al., Cancer Res 1999;59:6091-6). More recently, amplification and overexpression of the neighboring contactin 2 gene (CNTN2) was reported in individual malignant gliomas without MDM4 amplification (Rickman et al., Cancer Res 2001;61:2162-8). To address the question of whether 1q32 carries 2 independent amplification targets or a common target other than MDM4 and CNTN2, we analyzed primary malignant gliomas for amplification and overexpression of 17 different genes from this region. Our results indicate a single region of amplification that comprises the genes MDM4, GAC1, PIK3C2B and PEPP3, with only MDM4 amplification being invariably associated with overexpression. CNTN2 was found to be coamplified with MDM4 in 3 malignant gliomas but overexpressed in only 1 of these tumors. No CNTN2 amplification was detected in any of 102 malignant gliomas without MDM4 amplification. Our data therefore corroborate the notion that MDM4 is the main amplification target on 1q32 in malignant gliomas. However, coamplification and overexpression of adjacent genes may provide an additional growth advantage in some malignant gliomas with MDM4 amplification., (Copyright 2003 Wiley-Liss, Inc.)

To investigate the genetic mechanism of metastatic spread in hepatocellular carcinoma (HCC), we analyzed genomic changes in lung or liver metastases and the corresponding primary tumors (83 tumor samples) in 18 patients who underwent orthotopic liver transplantation. We studied the incidence of microsatellite instability (MSI) and loss of heterozygosity (LOH) involving 8 highly polymorphic microsatellite markers and the polyA tract, Bat26. We also sought alterations of p53 and beta-catenin gene mutations. High MSI (>30-40% of the loci analyzed) was found only in primary tumors (11%), whereas LOH was observed in 50% of primary and in 39% of recurrent tumors. p53 mutations were found in 2 cases of primary HCC but not in the corresponding metastases. P53 was overexpressed in 4 primary HCC (22%) and 7 metastases (39%). The percentage of beta-catenin gene mutations was low (6%). Lung metastases retained the D16S402 microsatellite abnormalities observed in the primary tumors, whereas recurrent liver tumor did not (p = 0.02). In conclusion, LOH and P53 protein overexpression, rather than mutations in the p53 or beta-catenin genes or MSI, seem to be involved in the spreading of HCC, suggesting the presence of metastasis suppressor genes in the vicinity of the chromosomal loci in question., (Copyright 2003 Wiley-Liss, Inc.)

Telomerase activity is detectable in most human tumors but not in most normal somatic cells or tissues. Telomerase inhibition has, therefore, been proposed as a novel and potentially selective strategy for antitumor therapy. In the present study, we found that platinum compounds, including cisplatin [cis-diamminedichloro-platinum (II)], strongly inhibited the activity of partially purified rat telomerase. Among the agents tested, 2,3-dibromosuccinato [2-(methylaminomethyl)pyridine]platinum (II) (compound E) exhibited the strongest inhibition, with an median inhibitory concentration (IC(50)) of 0.8 micro M. The mode of inhibition was noncompetitive with either dNTPs or TS (first) primer, with K(i) values estimated to be 2.3 or 3.9 micro M for varied TS primer or dNTPs, respectively. Notably, cisplatin also inhibited the telomerase activity, with an IC(50) of 2.0 micro M. Again, the mode of inhibition was noncompetitive, with K(i) values estimated as 7.3 or 8.1 micro M. Preincubation of TS primer with compound E did not affect the telomerase inhibition, whereas preincubation with cisplatin caused remarkable enhancement. Treatment of a human hepatoma cell line HepG2 with a low concentration of compound E gradually reduced the telomere length, indicating that this compound was able to inhibit telomerase in living cells as well as in vitro., (Copyright 2003 Wiley-Liss, Inc.)

Arsenic is well established as a human carcinogen, but its precise mechanism of action remains unknown. Arsenic does not directly damage DNA, but may act as a carcinogen through inhibition of DNA repair mechanisms, leading indirectly to increased mutations from other DNA damaging agents. The molecular mechanism underlying arsenic inhibition of nucleotide excision repair after UV irradiation (Hartwig et al., Carcinogenesis 1997;18:399-405) is unknown, but could be due to decreased expression of critical genes involved in nucleotide excision repair of damaged DNA. This hypothesis was tested by analyzing expression of repair genes and arsenic exposure in a subset of 16 individuals enrolled in a population based case-control study investigating arsenic exposure and cancer risk in New Hampshire. Toenail arsenic levels were inversely correlated with expression of critical members of the nucleotide excision repair complex, ERCC1 (r(2) = 0.82, p < 0.0001), XPF (r(2) = 0.56, p < 0.002), and XPB (r(2) = 0.75, p < 0.0001). The internal dose marker, toenail arsenic level, was more strongly associated with changes in expression of these genes than drinking water arsenic concentration. Our findings, based on human exposure to arsenic in a US population, show an association between biomarkers of arsenic exposure and expression of DNA repair genes. Although our findings need verification in a larger study group, they are consistent with the hypothesis that inhibition of DNA repair capacity is a potential mechanism for the co-carcinogenic activity of arsenic., (Copyright 2003 Wiley-Liss, Inc.)

Hereditary nonpolyposis colorectal cancer (HNPCC) is often caused by a deficiency in DNA mismatch repair. By using conventional methods of mutation analysis, point mutations in the DNA mismatch repair genes MSH2 and MLH1 have been detected in up to 64% of patients suspected of HNPCC. However, large genomic deletions cannot be detected by these methods. In our study, we applied a semiquantitative multiplex PCR to detect the proportion of large deletions in patients meeting the Bethesda criteria whose tumours exhibited microsatellite instability (MSI). Of 368 unrelated patients, 180 exhibited MSI. In these patients, 68 disease-causing point mutations (38%) had previously been detected in the MSH2 and MLH1 genes by SSCP, heteroduplex analysis or DHPLC followed by direct sequencing. The remaining 112 patients (including 24 patients with rare missense or other unclarified variants) were examined for large deletions. We identified deletions in 19 patients (10.6%); 11/19 (58%) deletions were located in MSH2 and 8/19 (42%) in MLH1, respectively. The size of deletions ranged from 1 exon to a deletion of a whole gene. Five breakpoints of deletions were sequenced; Alu-repetitive elements were involved in all of them. In patients meeting the Amsterdam criteria the proportion of large deletions was 12.6%. A similar proportion of deletions was found in the group of patients with a positive family history for colorectal cancer and MSI tumours, not meeting the Amsterdam criteria. The results of our study suggest that large genomic deletions in both MSH2 and MLH1 genes play a considerable role in the pathogenesis of HNPCC and should be part of the routine HNPCC mutation detection protocols., (Copyright 2002 Wiley-Liss, Inc.)

CPEC is a potent inhibitor of CTP synthetase and causes depletion of CTP and dCTP pools. AraC is an analog of dCyd and a chemotherapeutic agent. Here, we demonstrate that, upon incubation with CPEC, both the anabolism and cytostatic effect of AraC in SK-N-BE(2)c neuroblastoma cells were increased. Cotreatment of CPEC (50-250 nM) and AraC (37.5-500 nM) decreased the 4-day ED(50) value for AraC 2- to 8-fold in the SK-N-BE(2)c cell line, while pretreatment with CPEC followed by incubation with AraC alone decreased the 4-day ED(50) value for AraC 1- to 19-fold. Preincubation of SK-N-BE(2)c cells with 100 nM CPEC followed by incubation with 500 nM [(3)H]AraC increased the total amount of AraC nucleotides and incorporation of [(3)H]AraC into DNA by 392% and 337%, respectively, compared to non-CPEC-treated cells. When 20 nM [(3)H]AraC was used, the maximum incorporation of [(3)H]AraC into DNA was 1,378% compared to non-CPEC-treated cells. Incorporation of AraC into DNA correlated well with the accumulation of cells in S phase of the cell cycle caused by CPEC. DNA synthesis was almost completely inhibited (>91%) when 100 nM CPEC and 500 nM AraC were combined. CPEC alone and the combination of CPEC and AraC increased caspase-3 activity 3-fold, indicating induction of apoptosis in SK-N-BE(2)c cells. In contrast, AraC alone did not induce caspase-3 activity. Our results demonstrate that low concentrations of CPEC profoundly increase the cytostatic properties of AraC toward SK-N-BE(2)c human neuroblastoma cells., (Copyright 2002 Wiley-Liss, Inc.)

The causal role of human papillomavirus (HPV) in cervical carcinogenesis is beyond reasonable questioning. The progression from HPV infection, squamous intraepithelial lesions (SIL) to squamous cell carcinomata (SCC), however, is very uncommon and inefficient. Host genetic factors that may confer the susceptibility of disease progression are largely unknown. Apoptosis is an important fail-safe check for tumor development, in which Fas/FasL interaction contributes substantially. The purpose of our study is to test the hypothesis that an A/G polymorphism at -670 of Fas promoter with different transcriptional activity is associated with the risk for cervical neoplasia. A hospital-based case-control study was conducted, in which 104 patients of low grade SIL (LSIL), 131 high grade SIL (HSIL) and 176 SCC as well as age-matched, 1:1 controls were tested for Fas polymorphism by PCR-RFLP. HPV genotypes were determined in case groups by MY PCR-reverse line blot. The frequency of A allele was significantly (p = 0.006) higher in SCC than in control, conferring an odd ratio of 1.5 (95% CI = 1.1-2.0). The distribution of Fas (-670) genotypes also differed significantly between HSIL, SCC and each of their control (p = 0.017 and 0.03, respectively), with the A/A genotype conferring an OR of 1.3 (95% CI = 1.1-1.6) and 1.6 (95% CI = 1.0-2.5), respectively. Remarkably, the frequency of A allele and A/A genotype increased gradually in accordance with the multi-step carcinogenesis from LSIL, HSIL to SCC (p(test for trend) = 0.0066 and 0.0007, respectively). In addition, there was no difference of Fas genotypes between HPV (+) and HPV (-) cases. Fas genotypes, however, differed in LSIL infected with different HPV types (p = 0.033). The present study demonstrated an association between Fas polymorphism and cervical carcinogenesis. We deduced a possible effect of apoptosis of immune cells in this virus-induced cancer., (Copyright 2002 Wiley-Liss, Inc.)

The HER2 oncogene, which encodes the tyrosine kinase receptor, is commonly overexpressed in several types of cancer. Treatment using a humanized monoclonal antibody bound to HER2 product is becoming standard therapy for advanced breast cancer. Overexpression occurs in approximately 30% of non-small cell lung cancers (NSCLCs) and has been associated with poor prognosis. However, the frequency of a genetic aberration in the HER2 gene in lung cancer and the association between gene amplification and prognosis are poorly defined. To clarify these relationships, we simultaneously analyzed protein overexpression by immunohistochemistry (IHC) and determined the gene copy number by FISH in 50 surgical specimens of NSCLC. A low-grade increase in the copy number (3 to 8 copies) of the HER2 gene was detected in 44% of tumors. Most represented polysomy of chromosome 17. Protein overexpression was observed in 26%. Overexpression was detected in adenocarcinoma more frequently than in squamous cell carcinoma. No significant correlation was observed between copy number increase and overexpression. Neither gene copy number increase nor overexpression correlated with survival. We conclude that the significance of HER2 status in NSCLC is different from that in breast cancer because high-grade amplification occurs rarely., (Copyright 2002 Wiley-Liss, Inc.)

Aberrantly methylated DNA fragments in a human gastric cancer were searched for by a genome-scanning method, methylation-sensitive-representational difference analysis (MS-RDA). Six DNA fragments flanked by CpG islands (CGIs) and hypermethylated in the cancer were isolated. Four of the 6 fragments possessed genes in their vicinities. Quantitative RT-PCR analysis of the 4 genes showed reduced expression of 2 genes in cancers: Insulin-induced protein 1 (INSIG1/CL-6) and p41 Arp2/3 complex (p41-Arc). As for INSIG1, a DNA fragment was derived from the edge of a CGI in the promoter region. The edge was methylated in 11 of 22 primary gastric cancers, whereas the center was not methylated in any cancer. INSIG1 expression was markedly reduced in 19 cancers, including the 11 cancers with the methylation. By 5-aza-2'-deoxycytidine treatment of 5 cell lines with the methylation of the edge, partial restoration of INSIG1 expression was detected only in 2 of them. These data indicated that, although the reduced INSIG1 expression in cancers was associated with the methylation at the edge of the CGI in the promoter region, the methylation was likely to be a secondary change. As for p41-Arc, a DNA fragment was derived from a CGI overlapping exon 8, and its methylation did not correlate with its expression. However, methylation of a CGI in the promoter region with a marked reduction of its expression was observed in 1 of the 22 primary cancers. INSIG1 and p41-Arc are known to be involved in cellular differentiation and morphology, respectively, and it was suggested that their reduced expressions might be involved in gastric cancer development or progression., (Copyright 2002 Wiley-Liss, Inc.)

Chromosome 1 abnormalities are the most commonly detected aberrations in many cancers including malignant melanoma. Partial deletions and an allelic loss of the chromosome 1p36 region observed in melanoma indicate the presence of putative tumor suppressor gene(s) in this region. A candidate gene, CDC2L1, which encodes PITSLRE proteins related to p34(cdc2), is mapped to 1p36. To determine whether CDC2L1 mutation is involved in melanoma development, we examined 20 melanoma cell lines and 11 members of melanoma-prone families linked to chromosome 1p36. Mutation analysis throughout the entire coding region of the CDC2L1 gene revealed only 1 mutation (C-->T at nucleotide location 97 of exon 7, Ser-->Leu) in the melanoma cell line UACC 903 out of 20 melanoma cell lines and 6 melanoma cases. However, 4 polymorphic nucleotide changes, C-48T, G-53C, T-103C and T-210C, in the putative promoter region of CDC2L1 were identified. The 4 variants were located within or beside the conserved binding sites of transcription factors TCF11, MZF1 and TAAC box, indicating their potential effects on the regulation of CDC2L1 expression. No aberrant methylation of the CDC2L1 CpG island in the promoter region was observed by sodium bisulfite genomic sequencing. These results indicate that mutations are rare in the CDC2L1 gene in these melanoma cell lines and melanoma families and that the aberrant cytosine methylation of the CDC2L1 CpG island is not the mechanism of CDC2L1 repression in melanoma. The contribution of 4 promoter polymorphisms to the transcriptional regulation of the gene and its association with melanoma warrants further investigation., (Copyright 2002 Wiley-Liss, Inc.)

The development of cutaneous malignant melanoma (CMM) and its precursor lesions, melanocytic nevi, has been linked to sun exposure. Cyclobutane pyrimidine dimers (CPDs) are the majority of DNA lesions induced by sun exposure. In our study, we investigated if CMM patients have impaired ability to repair CPDs in skin as well as in melanocytic nevi. The repair kinetics were followed up to 3 weeks after exposure to 40 mJ/cm(2) of solar simulating radiation. Altogether 12 CMM patients and 10 healthy controls were included in our study. Buttock skin biopsies were taken at 0 hr, 48 hr and 3 weeks after UV exposure, whereas melanocytic nevi and surrounding skin biopsies were taken only at 0 hr and 3 weeks. The CPD levels were measured by a (32)P-postlabeling method. The results showed that the repair rate of CPDs in neither the skin nor the nevi was significantly different between the CMM patients and the control group. For both groups, the repair rate of TT = C was faster than that for TT = T. The important finding is that about 10% of the initial TT = T damage remained unrepaired after 3 weeks, and was detectable in normal epidermis as well as in nevi of all subjects. We also found that the amount of TT = C and TT = T at 0 hr in nevi was significantly lower than that in surrounding skin (Wilcoxon rank sum test, p < 0.05)., (Copyright 2002 Wiley-Liss, Inc.)