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PubMed2003

Molecular phenotype of spontaneously arising 4N (G2-tetraploid) intermediates of neoplastic progression in Barrett's esophagus.

Elevated 4N (G(2)-tetraploid) cell populations are unstable intermediates in the development of many human cancers. However, 4N cell populations are intermixed with larger diploid fractions in vivo, limiting investigation of these key intermediates of neoplastic progression. Therefore, to study elevated 4N cell populations in human neoplasia, we used flow cytometry to purify populations of spontaneously arising TP53(wt) and TP53(mut) 4N cells from cell strains derived from premalignant Barrett's esophagus biopsies. Using oligonucleotide arrays, we identified 625 genes differentially expressed in at least one replicate 2N/4N comparison in each strain and in hTERT-immortalized cultures of the TP53(mut) strains. Strikingly, when hierarchically clustered, these data contained a large node of 124 genes that were up-regulated in 4N TP53(mut) cells in the absence of condensed chromosomes. Most of these genes function in G(2)-M to mediate processes such as chromosome condensation and segregation. These results describe the molecular phenotype of dysregulated G(2)-M functions and cell cycle checkpoints in a key intermediate of human neoplastic progression.

Barrett Esophagus↗
PubMed2003

[Low efficiency of DNA repair system in mitochondria].

Under the action of endogenous reactive oxygen species and exogenous chemical and physical agents, significantly more lesions occur in mitochondrial DNA (mtDNA) than in nuclear DNA (nDNA). However, the mechanisms of DNA repair in mitochondria are less efficient that in the nuclei. The mechanisms of nucleotide excision repair capable of removing UV-induced lesions or other complex adducts induced by chemical compounds are not operative in mitochondria at all. At the same time, mitochondria of some kinds contain a photoreactivation enzyme providing monomerization of cyclobutane pyrimidine dimers. Also, the enzyme system for DNA base excision repair (BER) and O6-alkylguanine-DNA alkyl transferase are functional in mitochondria. However, the rate of BER-controlled repair of lesions in mtDNA is lower than that in nDNA. The literature data suggest that the controlling system for the delay of DNA replication till the repair complexion (cell cycle checkpoint) cannot be provided in mitochontria. Besides, it remains unclear whether the mismatch repair mechanisms are operable in mammalian mitochondria. On the other hand, double-strand breaks in mammalian mtDNA are possibly repaired by involving the DNA-dependent protein kinase complex, and the process of BER is affected by poly(ADP-ribosyl)ation of proteins. Possible consequences of induction of the increased level of damage in mtDNA and the low efficiency of repair systems in mitochondria are discussed in this review.

Animals↗
PubMed2003

The CHEK2*1100delC variant acts as a breast cancer risk modifier in non-BRCA1/BRCA2 multiple-case families.

The frame-shifting mutation 1100delC in the cell-cycle-checkpoint kinase 2 gene (CHEK2) has been reported to be associated with familial breast cancer in families in which mutations in BRCA1 and BRCA2 were excluded. To investigate the role of this variant as a candidate breast cancer susceptibility allele, we determined its prevalence in 237 breast cancer patients and 331 healthy relatives derived from 71 non-BRCA1/BRCA2 multiple-case early onset breast cancer families. Twenty-seven patients (11.4%) were carrying the CHEK2*1100delC variant. At least one carrier was found in 15 of the 71 families (21.1%). There was no evidence of cosegregation between the variant and breast cancer, but carrier patients developed breast cancer earlier than did noncarriers. We studied CHEK2 protein expression in 111, and loss of heterozygosity at CHEK2 in 88 breast tumors from these patients. Twelve of 15 tumors from carriers showed absent protein expression as opposed to 3 of 76 tumors from noncarriers (P < 0.001). CHEK2 loss of heterozygosity was associated with absence of protein expression but not with 1100delC carrier status. Thus, selecting for breast cancer cases with a strong familial background not accounted for by BRCA1 or BRCA2 strongly enriches for carriers of CHEK2*1100delC. Our results support a model in which CHEK2*1100delC interacts with an as yet unknown gene (or genes) to increase breast cancer risk.

Breast Neoplasms↗
PubMed2004

Nijmegen breakage syndrome and DNA double strand break repair by NBS1 complex.

The isolation of the NBS1 gene revealed the molecular mechanisms of DSB repair. In response to DNA damage, histone H2AX in the vicinity of DSBs is phosphorylated by ATM. NBS1 then targets the MRE11/RAD50 complex to the sites of DSBs through interaction of the FHA/BRCT domain with gamma-H2AX. NBSI complex binds to damaged-DNA directly, and HR repair is initiated. To collaborate DSB repair, ATM also regulates cell cycle checkpoints at GI, G2, and intra-S phases via phosphorylation of SMC, CHK2 and FANCD2. The phosphorylation of these proteins require NBS1 complex. Thus, NBSI has at least two important roles in genome maintenance, as a DNA repair protein in HR pathway and as a signal modifier in intra-S phase checkpoints. NBSI is also known to be involved in maintenance of telomores, which have DSB-like structures and defects here can cause telomcric fusion. Therefore, NBS1 should be a multi-functional protein for the maintenance of genomic integrity. Further studies on NBS1 will provide insights into the mechanisms of DNA damage response and the network of these factors involved in genomic stability.

Journal Article↗
PubMed2005

[Anti-proliferation effect of genistein on salivary adenoid cystic carcinoma cell line SACC-83 in vitro].

PURPOSE: To investigate the anti-proliferation effect of tyrosine protein kinase inhibitor, genistein (4'-5,7-trihydroxyisoflavone), on human salivary adenoid cystic carcinoma cell line SACC-83 in vitro, and its effect on cell cycle. METHODS: SACC-83 cells cultured in vitro were treated with genistein,cell survival rate was calculated with MTT assay, phase contrast microscope was used to observe the status of cell growth and the morphological changes in each group, and cell cycle was detected with flow cytometry. Apoptosis was detected with Annexin V/PI staining. The results were statistically analyzed by SPSS11.5 software. RESULTS: When treated with genistein of certain concentration for certain time, SACC-83 cell growths were significantly inhibited. With the increase of concentration of genistein and elongation of acting time, the inhibitory effects increased. Cells treated with genistein showed changes in morphology, cells' volume was decreased ,and suspended cells increased. Treated with 220 micromol/L genistein for 72 hours, SACC-83 cell growths were significantly inhibited, cell cycle was arrested in G2/M phase, and cell apoptosis was induced(P<0.01). CONCLUSION: Genistein inhibits growth of human salivary adenoid cystic carcinoma cell line SACC-83, arrests cell cycle in G2/M phase, and induces cell apoptosis; these suggest that tyrosine protein kinase has important effect on the neoplasia and development of salivary adenoid cystic carcinoma.

Apoptosis↗
PubMed2005

[The effect of gemcitabine on proliferation and telomerase activity of Tca8113 cell lines].

PURPOSE: To investigate the effect of gemcitabine on the proliferation and telomerase activity of Tca8113 cell line cells. METHODS: MTT assay, flow cytometry were used to examine the effect of gemcitabine on cell proliferation and cell cycle respectively, telomerase activity was detected with telomeric repeat amplification protocol (TRAP) and polyacrylamide gel electrophoresis (PAGE). The data were analyzed by independent samples t test and one-way ANOVA of SPSS10.0 for windows. RESULTS: Cell proliferations were significantly inhibited in these cells after exposure to gemcitabine as well as cell cycles were arrested. Furthermore, the effect was closely associated with the concentration and time of gemcitabine. The average rate of cell proliferating inhibition in group 5.0 microg/ml at 72 h was 72.2%.The number of cells in S phase was decreased,the average rate of S phase in group 5.0 microg/ml at 72 h was 18.1%,which was lower than that in group 0 microg/ml(38.5%) (t=5.801,P<0.05).Longer exposure time and higher concentration to gemcitabine, much telomerase activity was inhibited. After 72h, the A values of group 0 and 5.0 microg/ml were 1.32+/-0.12, 0.28+/-0.02, respectively (F=811.208, P<0.05). CONCLUSIONS: Gemcitabine could significantly inhibit Tca8113 proliferation and decrease its telomerase activity. Gemcitabine may be applied in clinical therapy of oral cancer.

Carcinoma, Squamous Cell↗
PubMed2006

Comparison of efficacy and toxicity profile between intraperitoneal and intravenous topotecan in human ovarian cancer xenografts.

OBJECTIVE: To compare the therapeutic and toxic profile of topotecan given intraperitoneally with intravenously in human ovarian cancer xenografted into athymic nude mice. METHOD: Eighty female Balb-c/nu-nu mice were randomized assigned into eight groups (n=10). Xenografts resulted from intramesentery injection of cultured human ovarian cancer cells SKOV3 in athymic mice. Onset of intraperitoneal treatment with either topotecan or cisplatin (7.5 mg/kg) was on day 7. Animals scheduled for topotecan i.p. received intraperitoneal application of topotecan (1.5 mg/kg x 2, 3.0 mg/kg x 2, 6.0 mg/kg x 2 or 10.0 mg/kg x 1). Animals scheduled for topotecan i.v. received intravenous administration of topotecan (6.0 mg/kg x 2 or 10.0 mg/kg x 1). Two weeks after drug application animals were killed. Tumor growth inhibition were assessed and compared with untreated mice and cisplatin intraperitoneally administered mice. Acute toxicity was determined by loss of body weight. Cell cycle division and apoptosis after drug administration was determined by flow cytometric analysis. RESULTS: In a panel of ten tumour xenografts, intraperitoneal topotecan was significantly more effective than intravenous administration. The toxicity profile suggested a better tolerability in terms of weight loss after intraperitoneal administration than cisplatin control. Topotecan 10.0 mg/kg i.p. per day (1 day) schedule was an optimal treatment for ovarian cancer and well tolerated by mice with no signs of acute toxicity. Topotecan and cisplatin induce cells G0-G1 arrest and apparent apoptosis. No significant difference among mice treated with topotecan intraperitoneally or intravenously or cisplatin was observed in term of apoptosis and cell cycle perturbation. CONCLUSION: The results may have implications for the future design of clinical studies on intraperitoneal application of topotecan. It suggests that apoptosis and cell cycle perturbation play an limited role in the mechanism of topotecan administration.

Animals↗
PubMed2006

Cigarette smoke extract inhibits the proliferation of alveolar epithelial cells and induces apoptosis.

Cigarette smoke extract (CSE) contains abundant oxidants and free radicals. Oxidative stress caused by cigarette smoking results in the destruction of the alveolar cell walls and emphysema. However, there exists discrepancy about how CSE works in the process. In the present study, we observed the effect of CSE on the cell growth of type II alveolar epithelial cell-derived A549 cell line, and provided molecular understanding of this effect. The MTT assay results showed that CSE decreased the cell viability of A549 cells in a dose- and time-dependent manner, and cell cycle was arrested in G(1)/S phase. Furthermore, CSE-induced apoptosis of A549 cells was verified by Hoechst 33258 staining, electron microscopy in morphology, and the appearance of DNA fragmentation and annexin V-FITC/propidium iodide (PI) staining assay at molecular level. It was found that CSE treatment resulted in the upregulation of Fas/APO-1 receptor and activation of caspase-3. CSE also initiated accumulation of intracellular reactive oxygen species, which was detected by laser confocal microscopy. Taken together, CSE could inhibit the cell growth and induce apoptosis of A549 cells through Fas receptor pathway. Oxidative stress caused by CSE may be the radical factor leading to apoptosis as well as cell growth inhibition in alveolar epithelial cells.

Apoptosis↗
PubMed2007

Structure-activity relationship of a glycolipid, sulfoquinovosyl diacylglycerol, with the DNA binding activity of p53.

The in vitro relationship between the human p53 DNA binding domain (p53 DBD) and glycolipids was investigated. We isolated the glycolipid fraction from spinach (Spinacia oleracea L.) and found that the fraction inhibited the double-stranded DNA (dsDNA) binding activity of p53 DBD. Since the fraction contained mainly three glycolipids, monogalactosyl diacylglycerol (MGDG), digalactosyl diacylglycerol (DGDG) and sulfoquinovosyl diacylglycerol (SQDG), and each glycolipid was purified using silica gel column chromatography. Purified SQDG inhibited the activity, however, purified MGDG and DGDG had no influence. In this study, we demonstrated the structure-function relationship between chemically synthetic SQDG and p53 DBD. The major action is probably dependent on the fatty acid effect, although SQDG was a much stronger inhibitor than the fatty acid alone present in SQDG. The inhibitory activity of SQDG was weakened by the R248A mutant of p53 DBD, suggesting that R248 in the dsDNA binding site of p53 must be important for the inhibitory activity of SQDG. SQDG binding to p53 DBD could be reversed with a non-ionic detergent, Nonidet P-40. This is the first study of a glycolipid, SQDG, acting as a dsDNA binding inhibitor of p53, and it could be considered that a SQDG-containing thylakoid membrane in plant chloroplasts might regulate the activity of p53 for cell division, cell cycle checkpoint and tumor suppression.

Computer Simulation↗
PubMedDate not supplied

The CHEK2 1100delC variant in Swedish colorectal cancer.

BACKGROUND: The cell cycle checkpoint kinase 2 (CHEK2) 1100delC variant has recently been identified at high frequency in families with both breast and colorectal cancer, suggesting the possible role of this variant in colorectal cancer predisposition. PATIENTS AND METHODS: To evaluate the role of CHEK2 ll00delC among Swedish colorectal cancer patients, the variant frequency was determined in 174 selected familial cases, 644 unselected cases and 760 controls, as well as in l8 families used in the genome-wide linkage analysis, where weak linkage was seen for the region harboring the CHEK2 gene. RESULTS: CHEK2 l100delC was found in 1.15% of familial and in 0.93% of unselected cases, compared to 0.66% of controls, showing no significant difference between groups. One out of 45 familial cases with a family history of breast cancer was shown to be a carrier. The variant was not identified in the 18 families included in the linkage analysis. CONCLUSION: The CHEK2 1100delC was not significantly increased in Swedish colorectal cancer patients, however, in order to determine the role of the variant in colorectal cancer families with the history of breast cancer a larger sample size is needed.

Case-Control Studies↗
PubMed1994

The p53-dependent gamma-ray response of GADD45.

Activation of the human GADD45 gene by ionizing radiation (IR) has previously been shown to be dependent on the tumor suppressor and transcription factor p53 (M. B. Kastan, et al., Cell 71: 587-597, 1992). Unlike GADD45, the response of other DNA damage-inducible genes to IR is not dependent on p53 based on the observation that induction in a panel of cell lines did not correlate with a normal p53 status; this included human GADD153, another member of the gadd (growth arrest and DNA damage inducible) group; MyD118, a gene related to GADD45; and the protooncogenes c-jun and c-fos. This p53-dependent response of GADD45 was further investigated in human cells with halogenated pyrimidines, which act as radiosensitizers when incorporated into cellular DNA. When cellular DNA contained halogenated pyrimidines such as iododeoxyuridine (IdUrd), GADD45 gamma-ray induction, as measured by increased mRNA, was enhanced. Rapid induction could be seen with doses as low as 0.5 Gy, and substitution with IdUrd resulted in an approximately 2-fold increase in induction over a wide dose range. This level of IdUrd substitution produced a similar fold increase in cellular radiosensitivity and has been shown previously (T. M. Kinsella et al., Int. J. Radiation Oncology Biol. Phys. 13: 733-739, 1987) to produce a similar fold increase in DNA strand breaks after IR. Considering that substitution with halogenated pyrimidines would be expected to have little effect on other cellular targets after IR, these experiments indicate that actual damage to DNA, primarily strand breaks, is a major signal for the activation of this p53-dependent pathway that is required for GADD45 induction and for activation of the G1 "checkpoint" cell cycle delay.

CCAAT-Enhancer-Binding Proteins↗
PubMed1997

p53-independent induction of p21WAF1/CIP1 expression in pericentral hepatocytes following carbon tetrachloride intoxication.

The cyclin-dependent kinase, proliferating cell nuclear antigen, and stress-activated protein kinase/c-jun NH2 terminal kinase inhibitor p21WAF1/CIP1 can induce G1 arrest, and its expression coincides with the cessation of replication in many systems. We examined expression of p21 during the early stages of carbon tetrachloride intoxication in the mouse liver and observed a dramatic increase in p21 RNA levels between 4 and 8 h after administration. p21 expression, visualized by in situ hybridization, is induced in pericentral hepatocytes before carbon tetrachloride-induced necrosis. Examination of c-fos and c-myc expression patterns confirm that these immediate-early genes are induced in similar regions of the mouse liver. p21 induction is not dependent on p53; we observed similar levels and localization of p21 in wild-type and p53 null animals. Immunohistochemical localization of p21 and CCAAT/enhancer-binding protein expression shows that p21 protein accumulation is limited to a subset of CCAAT/enhancer-binding protein-positive hepatocytes. A second peak of periportal and intermediate zone-specific p21 gene expression, appearing 1-2 days after injection, is also p53 independent and may represent cell cycle checkpoints or postmitotic growth arrest. Sporadic p21 expression was also detected in pairs of hepatocytes distributed throughout the liver acini in healthy animals. Together, these data suggest several roles for p21 in the liver in response to toxicity, regeneration, and growth inhibition.

Animals↗
PubMed1997

Allelic loss at BRCA1, BRCA2, and adjacent loci in relation to TP53 abnormality in breast cancer.

Cells with abnormal TP53 lose cell cycle checkpoints, resulting in genomic instability and neoplastic transformation. However, the evidence linking the tumor-specific targets of genomic alteration to an abnormal TP53 is limited. The present study tested the hypothesis that TP53 abnormalities are correlated with an increased frequency of deletion of breast cancer susceptibility loci (17q and 13q) in breast carcinomas. Tumors from 90 patients were examined for TP53 abnormality and loss of heterozygosity (LOH) at 11 loci on 17q (17q11.2-21) and 13q (13q12-14), including the loci for BRCA1 and BRCA2. A higher frequency of LOH was consistently found at 17q or 13q loci in tumors with an abnormal TP53. The increased LOH in relation to TP53 abnormality was statistically significant at the BRCA1, D17S588, and D13S267 loci (P < 0.05) but not at the locus for BRCA2 (P = 0.64). These observations imply a possible link between an abnormal TP53 and specific genomic deletions of breast cancer susceptibility loci, which may provide clues to the role of TP53 during breast tumorigenesis.

Adult↗
PubMed1998

White-ivory assay of Drosophila melanogaster under deficient repair conditions.

The prediction ability of a test to detect genotoxic activity may be increased, at least from a theoretical point of view, by carrying it out under deficient repair conditions. The white-ivory (w[i]) assay of Drosophila melanogaster is a somatic mutation and recombination test (SMART) that essentially differs from other SMARTs by the endpoints that can be detected. In this article, we study the consequences, with the w(i) assay, of the introduction of two mutations, mus201 and mei-41, which produce deficiency in two different repair mechanisms: the nucleotide excision repair system and in a G2/M cell-cycle checkpoint, respectively. Ten chemicals, previously classified as positive in the w(i) assay, have been assayed in both deficient repair conditions. As in the w/w+ and mwh/flr3 SMARTs, the results obtained with the w(i) assay show that the use of deficient repair strains does not improve the detection of genotoxic effects. However, the utilization of these deficient repair strains has been shown to be a useful tool in mechanistic studies. In fact, it seems that the nucleotide excision repair system mainly eliminates some spontaneous and chemically-induced damages involved in the reversion of w(i), whereas the repair system deficient in mei-41 flies is partly necessary to recover revertant w(i) spots.

ATP-Binding Cassette Transporters↗
PubMed1998

[Radiosensitivity of Saccharomyces yeasts and srm genes: effects of srm1 and srm5 mutations].

The presence in the cell genotype of srm1 and srm5 (cdc28-srm) mutations decreasing the spontaneous rho- mutability was shown to have no effect on the rates of spontaneous nuclear gene mutations and gamma-ray-induced mitotic recombination. Mutation cdc28-srm exerts a marked effect on cell sensitivity to the lethal action of ionizing radiation and on the appearance of homoplasmic segregants generated from heteroplasmic diploids. Additive interactions between mutations cdc28-srm and each of the rad6 and rad52 mutations were revealed by an analysis of double mutants with respect to their sensitivity to radiation. Mutation rad9 was epistatic with mutation cdc28-srm. These data agree with the idea that the p34CDC28 gene product is a target for the RAD9-dependent feedback control operating at the cell cycle checkpoints (checkpoint control) and ensuring an additional amount of time for premitotic repair of chromosomal DNA damage.

Alleles↗
PubMed1998

Alterations of p16-pRb pathway and chromosome locus 9p21-22 in sporadic invasive breast carcinomas.

The p16-pRb pathway represents a vital cell-cycle checkpoint. In the present study we investigated the alterations of this G1-phase protein pathway using immunohistochemical and molecular methods in a series of 55 breast carcinomas and correlated the findings with clinicopathological features of the patients. Furthermore, we examined its relationship with the status of the chromosomal region 9p21-22 performing a deletion map analysis because there are indications that, in addition to CDKN2 and MTS2/p15(INK4B) tumor suppressor genes (TSGs), this area harbors other TSG(s). Aberrant expression (Ab) of p16 and pRb was observed in 26 (47%) and 16 (29%) of the carcinomas, respectively. A statistical trend pointing out an inverse relationship between p16 and pRb expression was found (p = 0.079). Analysis of the region that encodes for p16 by deletion mapping, a PCR-based methylation assay and PCR-SSCP, revealed that deletions and transcriptional silencing by methylation might represent the main mechanisms of CDKN2/p16(INK4A) inactivation in breast carcinomas. The results of deletion mapping also suggest that another TSG(s) may reside at the 9p21-22 area particularly at the D9S162 loci and that co-deletion of this putative gene with CDKN2/p16(INK4A) may play a role in breast carcinogenesis. In addition, microsatellite instability (MI), a marker of replication error phenotype (RER+), was observed with a frequency of 16% in the area examined and was inversely related with loss of heterozygosity (LOH). Interestingly, most cases with MI at the region encoding for p16 were aggregated in a subgroup of breast carcinomas with no other obvious genetic and/or epigenetic CDKN2/p16(INK4A) alterations. We speculate that there is an additional mechanism of CDKN2/p16(INK4A) inactivation. The relationship of p16 protein level pRb, status, the p16-pRb combined immunoprofiles, and the microsatellite alterations detected at the 9p21-22 locus with the patients' clinicopathological parameters revealed two significant correlations: one between normal pRb expression and lymph node involvement (p = 0.0263), and the other between microsatellite alterations (LOH and or MI) and tumor size (p = 9.2 x 10(-3)). In view of the heterogenous nature of breast cancer, we suggest that in a significant proportion of breast carcinomas, deregulation of the p16-pRb pathway in association with another, as-yet unidentified, TSG(s) of the 9p21-22 region may play a role in initiating or progressing the oncogenic procedure, while in other subgroups, alternative molecules may play this role.

Adult↗