Showing papers in "Molecular Carcinogenesis in 2003"

Neonatal diethylstilbestrol exposure induces persistent elevation of c-fos expression and hypomethylation in its exon-4 in mouse uterus

Abstract: Perinatal exposure to diethylstilbestrol (DES) induces reproductive tract cancers later in life in both humans and animals. Because there is no clear evidence that perinatal DES exposure induces gene mutation, we proposed that perinatal DES exposure causes epigenetic methylation changes that result in persistent alterations in gene expression, leading to tumorigenesis. The proto-oncogene c-fos is one of the immediately induced genes in uterine epithelium after estrogen simulation and a key player in uterine carcinogenesis. Here, we investigated c-fos expression in mice neonatally exposed to DES (2 microg/pup/day on postnatal days 1-5). The mRNA levels of c-fos in uteri of neonatal DES-treated mice were persistently 1.4-1.9-fold higher than that in the control mice from day 5 to day 60. Overall, the uterine c-fos expression level in the neonatal DES-exposed group was significantly higher than that in the control group. After examination of the methylation status of the c-fos gene, we found that the CpGs in promoter and intron-1 regions were completely unmethylated. In exon-4, from day 17 to day 60, the percentage of unmethylated CpGs was higher in neonatal DES-exposed mice uteri than that in control (42%, 51%, 47%, and 42% in DES-exposed mice vs 33%, 34%, 33%, and 21% in control mice at day 17, 21, 30, and 60, respectively). These results suggest that perinatal DES exposure may permanently alter gene expression and methylation, and the methylation modification may occur in either the promoter regions or other regulatory sites in the gene.

Inhibition of cutaneous ultraviolet light B–mediated inflammation and tumor formation with topical celecoxib treatment

Abstract: Inflammation, which includes the release of growth factors, proinflammatory cytokines and prostaglandins, the infiltration and activation of inflammatory cells, and the induction of oxidative DNA damage, is known to play a role in cancer development. The combination of damage to the skin resulting from chronic ultraviolet light B (UVB) exposure itself and the inflammatory response it induces is a major source of skin cancer development. Cyclooxygenase-2 (COX-2), an inflammatory enzyme responsible for the production of prostaglandins, is now implicated in the development of epithelial cancers, including squamous cell carcinoma in the skin. Previous work conducted in our laboratory has shown that topical treatment with celecoxib following UVB irradiation inhibits several parameters of acute inflammation, including vascular permeability, the infiltration and activation of neutrophils, and the production of prostaglandin E(2) (PGE(2)). The present studies expanded these observations, demonstrating the ability of topical celecoxib to inhibit acute oxidative damage. In addition, long-term studies illustrate the effectiveness of topical treatment with this drug in reducing chronic inflammation and UVB-induced papilloma/carcinoma formation. This data provides compelling evidence to explore the clinical efficacy of topically applied COX-2 inhibitors for the prevention of human skin cancers.

Critical DNA damage recognition functions of XPC-hHR23B and XPA-RPA in nucleotide excision repair.

Abstract: It has been reported that 80-90% of human cancers may result, in part, from DNA damage. Cell survival depends critically on the stability of our DNA and exquisitely sensitive DNA repair mechanisms have developed as a result. In humans, nucleotide excision repair (NER) protects the DNA against the mutagenic effects of carcinogens and ultraviolet (UV) radiation from sun exposure. By preventing mutations from forming in the DNA, the repair machinery ultimately protects us from developing cancers. DNA damage recognition is the rate-limiting step in repair, and although many details of NER have been elucidated, the mechanisms by which DNA damage is recognized remain to be fully determined. Two primary protein complexes have been proposed as the damaged DNA recognition factor in NER: xeroderma pigmentosum protein A-replication protein A (XPA-RPA) and xeroderma pigmentosum protein C-human homolog of RAD23B (XPC-hHR23B). Here we compare the evidence that supports damage detection by these protein complexes and propose a model for DNA damage recognition in NER based on the current understanding of the roles these proteins may play in the processing of DNA lesions.

Hepatocyte growth factor, transforming growth factor alpha, and their receptors as combined markers of prognosis in hepatocellular carcinoma.

Abstract: A change in the balance between proliferation and apoptosis in the course of hepatocellular carcinoma (HCC) development and progression has been suspected. We wanted to identify related genes whose mRNA levels could provide markers of severity and prognosis after resection. The extent of cell apoptosis, proliferation, and differentiation was measured with a terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate-biotin nick-end labeling assay, and the Ki-67 index was determined in paired tumor and cirrhotic tissue samples from patients who had undergone HCC resection after diagnosis of hepatitis C-related or alcoholism-related cirrhosis. These patients included two groups with highly versus poorly differentiated tumor cells, and the latter was split into two subgroups of those with versus without early recurrence. The mRNA levels for various apoptosis-related or proliferation-related genes and those for the growth factor/receptor systems were measured by quantitative reverse transcriptase-polymerase chain reaction in paired tumor and cirrhotic liver samples from every patient, and some of the corresponding proteins were detected by immunohistochemistry. In all instances, protein expression was highly heterogeneous within groups and similar between groups. In contrast, some differences in mRNA level between tumor and cirrhotic tissues were quite informative. Low levels of hepatocyte growth factor and transforming growth factor alpha mRNAs were found concomitantly in highly differentiated tumors, whereas overexpression of mRNAs for the cognate receptors c-met and epidermal growth factor receptor were found in poorly differentiated tumors and primarily in patients with early tumor recurrence. These results argue for growth factor-dependent HCC development and provide novel and combined prognosis markers after HCC surgery.

Trichostatin A, an inhibitor of histone deacetylases, strongly suppresses growth of pancreatic adenocarcinoma cells.

Abstract: in cells with an altered p53 gene, the expression of p21 W A F 1 / C I P 1 , a potent inhibitor of cyclin-dependent kinases, can be induced by histone deacetylase (HDAC) inhibitors via a p53-independent pathway, which may play a critical role in arrest of cell growth. Accordingly, HDAC inhibitors such as trichostatin A (TSA) have potential utility in pancreatic cancer, as most of these tumors possess mutations in p53, which in fact is the main cause of chemoresistance to 5-fluorouracil. We have analyzed the effect of TSA on the proliferation of nine pancreatic adenocarcinoma cell lines, all containing a mutated p53 gene. TSA strongly inhibited the cellular growth of all these cell lines at submicromolar concentrations. The cellular mechanisms underlying this effect consisted of cell cycle arrest at the G2 phase and apoptotic cell death. The expression of p21 W A F 1 / C I P 1 normally induced at the transcriptional level by p53 was also strongly acivated by TSA. These findings suggest that inhibitors of HDAC may represent a novel therapeutic strategy for treatment of pancreatic cancer.

Molecular determinants of tumor differentiation in papillary serous ovarian carcinoma.

Abstract: In epithelial ovarian cancer, tumor grade is an independent prognosticator whose molecular determinants remain unknown. We investigated patterns of gene expression in well- and poorly differentiated serous papillary ovarian and peritoneal carcinomas with cDNA microarrays. A 6500-feature cDNA microarray was used for comparison of the molecular profiles of eight grade III and four grade I stage III serous papillary adenocarcinomas. With a modified F-test in conjunction with random permutations, 99 genes whose expression was significantly different between grade I and grade III tumors were identified (P < 0.01). A disproportionate number of these differentially expressed genes were located on the chromosomal regions 20q13 and all exhibited higher expression in grade III tumors. Interphase fluorescent in situ hybridization demonstrated 20q13 amplification in two of the four grade III and none of the three grade I tumors available for evaluation. Several centrosome-related genes also showed higher expression in grade III tumors. We propose a model in which tumor differentiation is inversely correlated with the overexpression of several oncogenes located on 20q13, a common amplicon in ovarian and numerous other cancers. Dysregulation of centrosome function is one potential mechanistic link between genetic/epigenetic changes and the poorly differentiated phenotype in ovarian cancer.

Polymorphisms of estrogen receptor alpha in prostate cancer

Abstract: Estrogen receptor (ER) alpha polymorphisms have been shown to be involved in the oncogenesis of several organs. We hypothesize that polymorphisms of the ERalpha gene are risk factors for prostate cancer. The genotypic distributions of six different loci (codons: 10 T-->C, 87 G-->C, 243 C-->T, 325 C-->G, 594 G-->A, and intron 1 C-->T) of the ERalpha gene were analyzed in prostate cancer tissues. The DNA from 115 cases of prostate cancer (Japanese population) was analyzed by sequence-specific polymerase chain reaction (PCR) and direct sequencing to determine the genotypic and allelic frequencies of the six different polymorphic loci of ERalpha. The relative risk of variant genotype was calculated by comparison with 200 healthy controls. Results of this study showed that the frequency of the variant genotype (C/C) on codon 10 was significantly higher in prostate cancer patients. The odds ratio (OR) was calculated as 3.26 compared to wild-type (T/T) with a 95% confidence interval (CI) of 1.58-6.73. Allele frequency at codon 10 also differed between groups. No association was found between codon 10 polymorphism and the stage of cancer. Polymorphism was not observed in codon 87, and frequencies of genotypes and alleles at other loci (intron 1, codons 243, 325, and 594) were not statistically different between cancer and controls. The present study suggests that polymorphism in codon 10 of ERalpha may be a risk factor for prostate cancer. These results are important in understanding the role of ERalpha polymorphism in the pathogenesis of prostate cancer.

Epigenetic inactivation of TSLC1 gene in nasopharyngeal carcinoma.

Abstract: Deletion of 11q23 is a common genetic aberration in nasopharyngeal carcinoma (NPC). Multiple candidate tumor suppressor genes (TSG) were mapped to this region but few of them were investigated in NPC. TSLC1 (tumor suppressor in lung cancer) is recently reported to be a putative TSG on 11q23. This gene was found to be inactivated by promoter hypermethylation in non-small cell lung carcinoma (NSCLC), liver cancer, and breast cancer. To study the role of TSLC1 gene in NPC tumorigenesis, we screened for mutations and aberrant methylation of TSLC1 gene in 5 NPC cell lines, 3 NPC xenografts, and 38 primary NPC cases. No somatic mutations of TSLC1 were detected in the NPC samples, but a 9-bp (CCACCACCA) deletion in exon 8 was found in a primary NPC and its corresponding blood sample. Bisulfite sequencing revealed aberrant methylation of TSLC1 promoter in four NPC cell lines. Loss of TSLC1 gene expression was found in two cell lines (HK-1 and CNE-2) with dense methylation. Expression of this gene was restored in these cell lines after treatment with demethylating agent 5-aza-2'-deoxycytidine. Our results showed that silencing of TSLC1 gene expression in NPC was associated with promoter hypermethylation. Promoter hypermethylation of TSLC1 gene was further illustrated in 34.2% (13/38) of primary NPCs. No aberrant promoter methylation was found in any of the four investigated normal nasopharyngeal epithelia. Frequent epigenetic inactivation of TSLC1 gene in NPC suggested that this gene is one of the target tumor suppressor genes of this endemic cancer.

EGF- and cell-cycle–regulated STAG1/PMEPA1/ERG1.2 belongs to a conserved gene family and is overexpressed and amplified in breast and ovarian cancer

Abstract: The abnormal activation of the epidermal growth factor (EGF) pathway is one of the most common findings in human cancer, and a number of molecular devices of laboratory and clinical relevance have been designed to block this transduction pathway. Because of the large number of cellular events that might be regulated through the activation of the four EGF receptor family members, it is possible that screening methodologies for the identification of new molecular targets working downstream of these pathways may provide new tools for cancer diagnosis and potentially prevention and therapy. In searching for EGF target genes, we have identified ERG1.2, the mouse homolog of the solid tumor-associated gene STAG1. Both in humans and in mice, it belongs to a new gene family that can give origin to several protein isoforms through alternative splicing and/or multiple translation starts. Sequence analysis and experimental data suggest that ERG1.2 is likely to function as a membrane-bound protein interacting with downstream signaling molecules through WW- and SH3-binding domains. ERG1.2 is a cell-cycle–regulated gene, and both ERG1.2 and STAG1 are induced by EGF and other growth factors at the transcript and protein levels. Finally, we have demonstrated that, besides prostate cancer and renal cell carcinoma, STAG1 was also overexpressed in breast and ovarian cancer cell lines and in breast primary tumors. Although in most cases STAG1 overexpression is probably due to the abnormal activation of the EGF pathway, we have also demonstrated genetic amplification and rearrangement of its locus in one breast cancer cell line and one primary ovarian cancer, suggesting that STAG1 might be a direct molecular target in the carcinogenetic process. Thus its overexpression might be regarded not only as a tumor marker but also as a potentially pathogenetic event. © 2003 Wiley-Liss, Inc.

TP53 is frequently altered by methylation, mutation, and/or deletion in acute lymphoblastic leukaemia.

Abstract: Different mechanisms, such as chromosomal rearrangements, deletions, mutations, and methylation/demethylation of the promoter regions of genes, have been shown to be involved in acute lymphoblastic leukaemia (ALL). These genetic and epigenetic alterations lead to the activation of protooncogenes or to inactivation of tumour supressor genes promoting cell proliferation. One of the most frequently inactivated tumour supressor genes is TP53, which is altered in 50% of human tumours. In this study, we have analysed: (1) the complete coding region, all intron-exon junctions and noncoding regions of exons 1–11 of TP53 by lexon-DGGE; (2) the methylation status of the 5′ region of TP53 and (3) the deletion of one or both alleles of the gene by fluorescence in situ hybridisation (FISH) in 57 ALL patients. Using these techniques, we have found promoter methylation in 32% of the cases, missense mutations in 8.8%, and deletion of one allele in 7.5% of the samples, with TP53 being altered in 40% of the ALL samples studied in this series. © 2003 Wiley-Liss, Inc.

Alsterpaullone, a novel cyclin-dependent kinase inhibitor, induces apoptosis by activation of caspase-9 due to perturbation in mitochondrial membrane potential.

Abstract: The majority of human neoplasms have aberrations in the retinoblastoma pathway due to hyperactivation of cyclin-dependent kinases (CDK). Based on this observation, novel small molecules, such as flavopiridol and UCN-01, are being developed and are currently being tested in the clinic. Efforts to develop CDK modulators led us to the discovery of a novel class of CDK inhibitors, the paullones [Cancer Res 1999;59:2566]. Initial studies demonstrated that paullones inhibit CDKs in vitro, thereby blocking cell-cycle progression. However, the exact mechanism for the antiproliferative effects of paullones was never explored. In this report, we demonstrate for the first time that the most potent paullone, alsterpaullone (Alp), induced apoptosis and promoted loss in clonogenicity in the Jurkat cell line. Alp caused early activation of both caspase-8 and -9, leading to cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP). Moreover, apoptosis by Alp was not associated with loss in anti-apoptotic proteins such as XIAP or BCL-XL. Pre-incubation with cell-permeable inhibitors z-Asp(OMe)-Glu(OMe)-Val-Asp(Ome)-fluoromethylketone and benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethylketone (ZVAD) blocked Alp-induced apoptosis. Moreover, the general caspase inhibitor ZVAD blocked the cleavage and activation of most caspases tested except caspase-9. Studies of mitochondrial membrane potential also demonstrated that Alp is able to disrupt mitochondrial potential in the presence of ZVAD, suggesting that the activation of caspase-9 by Alp follows mitochondrial perturbation. Pre-incubation of Jurkat cells with ZVAD did not prevent the depletion of cyclin D3, loss of CDK, or cell-cycle arrest by Alp. In summary, these experiments suggest that Alp activates caspase-9 via mitochondrial perturbation. Active caspase-9 cleaves and activates caspase-8 and caspase-3, leading to apoptosis. In the presence of the general caspase inhibitor ZVAD, the cell-cycle effects of Alp are unaltered while apoptosis is blocked, suggesting that the CDK effects of Alp are not sufficient for Alp-induced apoptosis. Additional studies with paullones are warranted to further characterize their preclinical effects and to explore their potential use in the clinical setting. Published 2003 Wiley-Liss, Inc.

Sequestration of connexin43 in the early endosomes: an early event of Leydig cell tumor progression.

Abstract: Connexins form gap junction channels that allow intercellular communication between neighboring cells. Compelling evidence has revealed that Cx are tumor-suppressor genes and reduced Cx expression has been related with uncontrolled cell growth in tumors and transformed cells. In the present study, we addressed Cx transcriptional and posttranscriptional regulations during the earlier stage of testicular tumors confined to Leydig cells in a transgenic mice model. In situ hybridization indicated that connexin43 (Cx43) mRNA was highly expressed either at early tumorogenesis (3 m) characterized by intense proliferation of Leydig cells, or at advanced tumorogenesis (6-7 m) when tumor cells completely invaded the testis. In contrast, Cx43 protein analyzed by Western blotting or classic immunohistochemical analyses was present at the beginning of tumor progression, but was dramatically reduced as tumor advanced. Application of high-resolution deconvolution microscopy to testis sections demonstrates that cells that proliferate exhibited an aberrant cytoplasmic Cx43 localization, in contrast to the expected plasma membrane Cx43 localization in normal Leydig cells. Dual immunofluorescence labeling with specific markers of cellular compartments shows that cytoplasmic Cx43 signal was mainly sequestered within early endosomes. Altogether, this study provides the first evidence that impaired Cx43 trafficking in endosomes is an early event associated with uncontrolled cell proliferation that could serve as a neoplastic marker.

Molecular mechanisms of G0/G1 cell-cycle arrest and apoptosis induced by terfenadine in human cancer cells.

Abstract: Terfenadine (TF), a highly potent histamine H1 receptor antagonist, has been shown to exert no significant central nervous system side effects in clinically effective doses. In this study, we demonstrated that TF induced significant growth inhibition of human cancer cells, including Hep G2, HT 29, and COLO 205 cells, through induction of G(0)/G(1) phase cell-cycle arrest. The minimal dose of TF induced significant G(0)/G(1) arrest in these cells was 1-3 microM. The protein levels of p53, p21/Cip1, and p27/Kip1 were significantly elevated, whereas the kinase activities of cyclin-dependent kinase 2 (CDK2) and CDK4 were inhibited simultaneously in the TF-treated cells. On the other hand, significant apoptosis, but not G(0)/G(1) arrest, was induced in the HL 60 (p53-null) or Hep 3B (with deleted p53) cells when treated with TF (3-5 microM). To clarify the roles of p21/Cip1 and p27/Kip1 protein expression, which was involved in G(0)/G(1) arrest and apoptosis induced by TF in human cancer cells, antisense oligodeoxynucleotides (ODNs) specific to p21/Cip1 and p27/Kip1 were used, and the expression of the p21/Cip1 and p27/Kip1 were monitored by immunoblotting analysis. Our data demonstrated that the percentage of the apoptotic cells detected by annexin V/PI analysis in the TF-treated group was clearly attenuated by pretreatment with p27/Kip1-specific ODNs. These results indicated that p27/Kip1 (but not p21/Cip1) protein indeed played a critical role in the TF-induced apoptosis. We also demonstrated that the TF-induced G(0)/G(1) cell-cycle arrest effect was not reversed by TF removal, and this growth inhibition lasted for at least 7 d. Importantly, the occurrence of apoptosis and cell growth arrest was not observed in the TF-treated normal human fibroblast, even at a dose as high as 25 microM. Our study showed the molecular mechanisms for TF-induced cell growth inhibition and the occurrence of apoptosis in human cancer cells.

Identification of distinct and common gene expression changes after oxidative stress and gamma and ultraviolet radiation.

Abstract: The human genome is exposed to many different kinds of DNA-damaging agents While most damage is detected and repaired through complex damage recognition and repair machineries, some damage has the potential to escape these mechanisms Unrepaired DNA damage can give rise to alterations and mutations in the genome in an individual cell, which can result in malignant transformation, especially when critical genes are deregulated In this study, we investigated gene expression changes in response to oxidative stress, gamma (gamma) radiation, and ultraviolet (UV) radiation and their potential implications in cancer development Doses were selected for each of the three treatments, based on their ability to cause a similar G(1) checkpoint induction and slow down in early S-phase progression, as reflected by a comparable reduction in cyclin E-associated kinase activity of at least 75% in logarithmically growing human dermal diploid fibroblasts To investigate gene expression changes, logarithmically growing dermal diploid fibroblasts were exposed to either gamma radiation (5 Gy), oxidative stress (75 microM of tert-butyl hydroperoxide (t-butyl-OOH)), or UV radiation (UVC) (75 J/m(2)) and RNA was harvested 6 h after treatment Gene expression was analyzed using the NIEHS Human ToxChip 20 with approximately 1901 cDNA clones representing known genes and expressed sequence tags (ESTs) We were able to identify common and distinct responses in dermal diploid fibroblasts to the three different stimuli used Within our analysis, gene expression profiles in response to gamma radiation and oxidative stress appeared to be more similar than profiles expressed after UV radiation Interestingly, equivalent cyclin E-associated kinase activity reduction with all the three treatments was associated with greater transcriptional changes after UV radiation than after gamma radiation and oxidative stress While samples treated with UV radiation displayed modulations of their mitogen activated protein kinase (MAPK) pathway, gamma radiation had its major influence on cell-cycle progression in S-phase and mitosis In addition, cell cultures from different individuals displayed significant differences in their gene expression responses to DNA damage

DNA repair gene O6-methylguanine-DNA methyltransferase: promoter hypermethylation associated with decreased expression and G:C to A:T mutations of p53 in brain tumors.

Abstract: The DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) removes alkylating adducts from the O6 position of guanine and protects cells from cytotoxic and mutagenic effects. Expression of MGMT is decreased in some cancers, which may be the result of methylation of CpG islands of both the promoter and coding regions of the gene. We studied the methylation status of the MGMT promoter in a very large collection of brain tumors (85) using methylation-specific polymerase chain reaction (PCR). Aberrant methylation occurred in 48% of 85 human brain tumor samples. Quantitative real-time PCR showed that expression of MGMT mRNA levels was significantly decreased (P < 0.001) in those brain tumors that had methylation of the promoter region of their MGMT gene. MGMT can prevent G to A mutations by removing alkyl groups from the O6 position of guanine. We found a significantly increased frequency of G:C to A:T mutations of the p53 gene in brain tumors having a methylated MGMT promoter compared with those having an unmethylated MGMT promoter (P < 0.05), and all the non–CpG dinucleotide G:C to A:T mutations of p53 were in samples with a methylated MGMT promoter. © 2002 Wiley-Liss, Inc.

Involvement of the Akt/mTOR pathway on EGF‐induced cell transformation

Abstract: Our previous study demonstrated that phosphatidylinositol 3-kinase (PI3K) is necessary for epidermal growth factor (EGF)-induced cell transformation in mouse epidermal JB6 cells. Akt and the mammalian target of rapamycin (mTOR) are regarded as PI3K downstream effectors. Therefore, in this study, we investigated the role of Akt and mTOR on EGF-induced cell transformation in JB6 cells using rapamycin, a specific mTOR inhibitor, and cells expressing dominant negative mutants of Akt1 (DNM-Akt1). We found that the treatment of cells with rapamycin inhibited EGF-induced cell transformation but only slightly inhibited JB6 cell proliferation at 72 h. Although LY294002, a PI3K inhibitor, attenuated EGF-induced activator protein 1 (AP-1) activation, treatment with rapamycin did not affect AP-1 activity. Treatment with rapamycin inhibited EGF-induced phosphorylation and activation of ribosomal p70 S6 protein kinase (p70 S6K), an mTOR downstream target, but had no effect on phosphorylation and activation of Akt. Rapamycin also had no effect on EGF-induced phosphorylation of extracellular signal-regulated protein kinases (ERKs). We showed that introduction of DNM-Akt1 into JB6 mouse epidermal Cl 41 (JB6 Cl 41) cells inhibits EGF-induced cell transformation without blocking cell proliferation. The expression of DNM-Akt1 also suppressed EGF-induced p70 S6K activation as well as Akt activation. These results indicated an involvement of the Akt/mTOR pathway in EGF-induced cell transformation in JB6 cells.

Chemotherapeutic efficacy of topical celecoxib in a murine model of ultraviolet light B-induced skin cancer.

Abstract: Over a million nonmelanoma skin cancer cases will be reported in the United States this year alone. Currently the primary form of treatment for these types of skin tumors is excision. However, excision of the initial lesion may not be curative because almost 50% of patients with one nonmelanoma skin cancer lesion develop another tumor within the next 5 yr at the site or adjacent to the site of excision. As with other types of epithelial based cancers, there is mounting evidence for the role of cyclooxygenase-2 (COX-2) and its products, particularly prostaglandin E2 (PGE2), in the development of nonmelanoma skin cancer. To avoid the excision process, the present study was designed to evaluate the possible chemotherapeutic effect of directly treating established tumors with a topical formulation of the specific COX-2 inhibitor celecoxib. Skh/hr hairless mice were irradiated three times per wk for 16 wk to induce tumor formation. The mice were then divided into two groups and treated topically with either 500 μg celecoxib or the vehicle for 6 wk. Our results demonstrated that although topical treatment with celecoxib was not able to induce regression of established tumors, it did prevent new tumor formation after the onset of photocarcinogenesis. Although further studies are warranted, these data suggest that topical celecoxib treatment may prove to be effective in preventing the recurrence of tumors at the site of nonmelanoma skin cancer excision. © 2003 Wiley-Liss, Inc.

Inhibition of cell-cycle effectors of proliferation in bladder tumor epithelial cells by the p75NTR tumor suppressor.

Abstract: The neurotrophin (NTR) receptor (p75(NTR)) is a cell-surface glycoprotein that binds to the neurotrophin family of growth factors, of which the prototypic member is nerve growth factor (NGF). This receptor was previously shown to retard cell-cycle progression by inducing accumulation of cells in G(1) with a concomitant reduction of cells in the S phase of the cell cycle. Furthermore, p75(NTR) was shown to be an effective tumor suppressor of bladder cancer cell growth in vivo. In order to investigate the mechanism of p75(NTR)-dependent suppression of cell-cycle progression, we utilized transgenic clones of bladder tumor cells that express p75(NTR) in increasing concentrations to demonstrate an effect of p75(NTR) on the levels of cell-cycle regulatory proteins that modulate proliferation of tumor cells. A rank-order (dose-dependent) increase in p75(NTR) protein expression was associated with a decrease in cell proliferation. This p75(NTR)-dependent suppression of proliferation was rescued with NGF. In the absence of ligand, a dose-dependent increase in p75(NTR) protein expression was associated with reduced expression of cyclin D1, cyclin E, and cyclin-dependent kinase 2 (cdk2) as well as decreased cdk2 activity. There was also a decrease in the expression of hyper-phosphorylated retinoblastoma protein, the transcription factor E2F1, and proliferating cell nuclear antigen, and there was an increase in expression of hypophosphorylated Rb and the cdk inhibitor p16(Ink4a) with increasing p75(NTR) expression. Treatment of tumor cells with NGF ameliorated these p75(NTR)-dependent changes in the levels of cell-cycle regulatory proteins and rescued the tumor cells from p75(NTR)-dependent inhibition of proliferation. Hence, it can be concluded that p75(NTR) inhibits proliferation by altering the expression of cell-cycle regulatory proteins and that NGF ameliorates this effect.

Microarray analysis of xenograft-derived cancer cell lines representing multiple experimental models of human prostate cancer

Abstract: Expression analysis of 7129 transcripts was carried out in five human prostate cancer cell lines derived from orthotopic xenografts after one to five passages in nude mice and primary cultures of human normal prostate epithelial (NPE) cells. These experiments identified a consensus class of 214 genes (43 up- and 171 downregulated transcripts), expression of which was altered at least twofold in the same direction in all the cell lines relative to NPE cells. To validate the relevance of altered expression behavior of these genes for human prostate cancer, their expression pattern was evaluated in multiple additional experimental and clinical settings. Expression of 170 of these 214 genes (79%) was altered in the same direction in vivo in experimental human prostate tumors in mice. Similarly, the expression of 151 of the 214 genes (71%) was altered in the same direction in M12 cells, a variant of an SV40 large T antigen transformed normal human prostate epithelial cell line selected for increased malignancy in vivo. In clinical samples of human prostate tumors, the changes in transcript expression levels of majority of these genes (85% of downregulated and 76% of upregulated transcripts) are consistent with alterations of their expression pattern in xenograft-derived cancer cell lines. These results imply that the expression pattern of a large class of genes is consistently altered in multiple experimental models and clinical samples of human prostate cancer and underscore the potential relevance of the xenograft models and cell lines derived from them for expression analysis studies relevant to human cancer.

Modulation of the angiogenesis response through Ha-ras control, placenta growth factor, and angiopoietin expression in mouse skin carcinogenesis.

Abstract: Tumor angiogenesis is governed by a complex balance of positive and negative angiogenic factors. Development of chemically-induced mouse skin tumors appears to be highly dependent on an early burst of neovascularization. We have previously shown that Ha-ras–driven vascular endothelial growth factor (VEGF) expression plays a pivotal role in this process. However, the status of other critical positive and negative angiogenic factors throughout skin tumorigenesis has not been studied to the same extent. In the present study, we show that another VEGF family member, placenta growth factor (PlGF), was highly upregulated at all tumor stages in a ras-dependent manner. The study of angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2), ligands of receptor tyrosine kinase 2 (Tie-2), showed that while stroma–derived Ang-2 was increased, epidermal Ang-1 expression was completely abolished at early papilloma formation. Studies using epidermal tumor cell lines suggest that the disappearance of Ang-1 also depends on ras activation, extending the plethora of events controlled by this oncogene in mouse skin carcinogenesis. Our results indicated that tumor development occurred in a strong angiogenesis-prone scenario in which PlGF and Ang-2 acted cooperatively with VEGF, whereas the negative or stabilizing effect of Ang-1 was abrogated. A time-course sequence of expression of angiogenic factors expressed throughout tumor growth, as well as the identification of key signaling molecules triggering the angiogenic response, may contribute to the development and testing of antiangiogenic therapeutic strategies with this in vivo tumor model. © 2003 Wiley-Liss, Inc.

Reduced gap junctional intercellular communication and altered biological effects in mouse osteoblast and rat liver oval cell lines transfected with dominant-negative connexin 43

Abstract: Gap junctional intercellular communication (GJIC) maintains normal growth and differentiation of cells in a tissue. The intercellular molecules traversing gap junctions are largely unknown, but the molecular weight (MW) cutoff is normally 1200 Da. No differences in dye transfer were observed in normal or vector controls of WB-F344 rat liver epithelial or mouse osteoblastic MC3T3-E1 cells with either Lucifer Yellow (LY) with a MW of 457 Da (LY-457) or LY with a MW of 649 Da (LY-649). Transfection of a dominant negative-connexin 43 (Cx43) gene decreased GJIC (>50%) when LY-649 was used, however, normal GJIC was observed in both cell lines when LY-457 was used. Therefore, the MW cut off in these clones was considerably less than the wild type. The dominant negative clones of the MC3T3-E1 cells exhibited over 90% less alkaline phosphatase (ALPase) activity and calcium deposition after the induction of differentiation. Similarly, dominant negative Cx43 inhibited gene expression of ALPase and bone sialoprotein but not osteocalcin in MC3T3-E1. WB-F344 cells normally exhibit a biphasic response to 12-O-tetradecanoylphorbol-13-acetate (TPA) where inhibition of GJIC recovers after 2 h, but the dominant negative clones showed no recovery from inhibition of GJIC by TPA. Dominant negative Cx43 also inhibited the formation of network-like structures by WB-F344 cells on Matrigel. These results demonstrate that the dominant negative gene transfected into cell types containing the wild-type connexins result in diminished channel sizes, thus allowing the determination of whether distinct biological endpoints, i.e., differentiation, are dependent upon either small or high MW intercellular signals.

Inhibition of proteasome‐dependent degradation of Wee1 in G2‐arrested Hep3B cells by TGFβ1

Abstract: Transforming growth factor β1 (TGFβ1)-induced G 2 arrest was observed when a proliferation inhibitory function of the retinoblastoma protein (Rb) was compromised, but the mechanism underlying the G 2 arrest was poorly characterized compared with that of G 1 arrest. In the present study, we characterized G 2 arrest induced by TGFβ1 (1 ng/mL) in the Rb-negative hepatoma cell line (Hep3B) and compared with G 1 arrest in the Rb-positive hepatoma cell line (Huh7). Activities of cyclin-dependent kinases (CDK) 2 and cell division cycle (CDC) 2 were markedly decreased at 24 h, the time when cell-cycle arrest became apparent in both cell lines. However, considerable amounts of inactive CDC2-cyclinB1 complexes were present in the nucleus of G 2 -arrested Hep3B but were not present in G 1 -arrested Huh7. The inhibitory phosphorylation of CDC2 on Tyr-15 was significantly elevated at 12-24 h, and its levels gradually declined during G 2 arrest in Hep3B. In particular, augmentation of CDK inhibitors p21 c i p 1 and p27 k i p 1 and Wee1 kinase and diminution of CDC25C phosphatase coincided with induced Tyr-15 phosphorylation and inhibition of CDC2. Wee1 in Hep3B was unstable and was degraded in a proteasome-dependent manner, but it became substantially stabilized within 6 h of TGFβ1 treatment. Moreover, a Wee1 inhibitor, PD0166285, abrogated the TGFβ1-induced G 2 arrest in Hep3B. These findings suggest that TGFβ1 induced G 2 arrest in Hep3B at least in part through stabilization of Wee1 and subsequent increase in Tyr-15 phosphorylation and inhibition of CDC2.

Chromosomal abnormalities in human glioblastomas: gain in chromosome 7p correlating with loss in chromosome 10q

Abstract: Various genomic alterations have been detected in glioblastoma Chromosome 7p, with the epidermal growth factor receptor locus, together with chromosome 10q, with the phosphatase and tensin homologue deleted in chromosome 10 and deleted in malignant brain tumors-1 loci, and chromosome 9p, with the cyclin-dependent kinase inhibitor 2A locus, are among the most frequently damaged chromosomal regions in glioblastoma In this study, we evaluated the genetic status of 32 glioblastomas by comparative genomic hybridization; the sensitivity of comparative genomic hybridization versus differential polymerase chain reaction to detect deletions at the phosphatase and tensin homologue deleted in chromosome 10, deleted in malignant brain tumors-1, and cyclin-dependent kinase inhibitor 2A loci and amplifications at the cyclin-dependent kinase 4 locus; the frequency of genetic lesions (gain or loss) at 16 different selected loci (including oncogenes, tumor-suppressor genes, and proliferation markers) mapping on 13 different chromosomes; and the possible existence of a statistical association between any pair of molecular markers studied, to subdivide the glioblastoma entity molecularly Comparative genomic hybridization showed that the most frequent region of gain was chromosome 7p, whereas the most frequent losses occurred on chromosomes 10q and 13q The only statistically significant association was found for 7p gain and 10q loss

P53 is a regulator of the metastasis suppressor gene Nm23-H1.

Abstract: p53, a tumor suppressor gene involved in the G1 cell cycle checkpoint, is also the most frequently mutated gene in human cancer. In addition, p53 modifies the ability of tumor cells to metastasize. The metastasis-associated gene Nm23-H1, which encodes an 18-kDa nucleoside diphosphate kinase, was previously identified in cells with low metastatic potential. Although p53 and Nm23-H1 proteins play an important part in regulating the progression of cancer, any functional relationship between these two proteins is currently unknown. Here we report an association between p53 levels and expression of the Nm23-H1 gene. Our data indicate that wild-type (wt) p53 upregulated the expression of Nm23-H1 at protein and mRNA levels in MCF-7 and J7B cells. This capacity of wt p53 to regulate expression of Nm23-H1 was not only dependent on the endogenous but also the exogenous origin of p53, and could not be reproduced with mutant p53. Subsequently, the invasive ability of MCF-7 and J7B cells was suppressed upon induction of the Nm23-H1 protein by p53. In contrast, increased levels of p53 downregulated the expression of Nm23-H1 at the protein and mRNA levels in RKO and H1299 cells and, as a consequence, increased the invasive ability of both cell types. Thus, our results implicated the differential regulation of Nm23-H1 by p53 in different cell types as an important component in the molecular mechanisms of tumor metastasis.

Cell growth inhibition by all-trans retinoic acid in SKBR-3 breast cancer cells: Involvement of protein kinase Cα and extracellular signal-regulated kinase mitogen-activated protein kinase

Abstract: All-trans retinoic acid (ATRA), a synthetic derivative of vitamin A, inhibits the growth of breast cancer cells. To elucidate the mechanism by which ATRA causes cell growth inhibition, we examined changes in cell cycle and intracellular signaling pathways, focusing on protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). Using the estrogen receptor-negative, retinoid receptor–positive breast cancer cell line SKRB-3, we found that treatment with ATRA significantly decreased the expression of PKCα, as well as reducing ERK MAPK phosphorylation. ATRA treatment leads to dephosphorylation of Rb, and consequently to G1 arrest. Marked changes in the expression of cyclins (particularly cyclins A and E) were observed in SKBR-3 cells treated with ATRA. Using a series of pharmacological and molecular approaches, we found evidence that ATRA-induced SKBR-3 cell growth inhibition involves the deregulation of the PKCα-MAPK pathway. These data suggest that retinoids interfered with signal transduction pathways that are crucial for cell cycle progression, and highlight the complexities of the biological effects of retinoid derivatives. © 2003 Wiley-Liss, Inc.

Tnfa and Il-10 deficiencies have contrasting effects on lung tumor susceptibility: gender-dependent modulation of IL-10 haploinsufficiency.

Abstract: Epidemiologic evidence suggests that pulmonary diseases with a prominent chronic inflammatory component elevate lung cancer risk. Genetic manipulations of mouse models of lung inflammation and tumorigenesis can be used to investigate this association. The genes encoding pro-inflammatory tumor necrosis factor-alpha (TNFalpha) and antiinflammatory IL-10 cytokines map within quantitative trait loci that regulate susceptibility to lung tumor development in mice; sensitive A/J and resistant C57BL/6J (B6) mice have different Tnfa and Il-10 alleles. Genetic ablation studies were performed to examine whether these genes would qualify as candidate tumor modifiers. Tnfa null (-/-) mice on a B6 background and B6.129 Il-10(-/-) mice were intercrossed with A/J mice and subjected to urethane carcinogenesis; lung tumor multiplicity was determined 20 weeks later. In the absence of one copy of Tnfa, tumor number. Male Il-10(+/+) mice developed more tumors than did female mice (P < 0.001), absence of one copy of Il-10 raised tumor number in female mice to that observed in +/+ males, but no change in multiplicity occurred in Il-10 hemizygous males. Thus, a deficit of pro-inflammatory TNFalpha decreased the number of tumors, whereas diminished gene copy number of anti-inflammatory IL-10 increased tumorigenesis; manifestation of an effect of Il-10 haploinsufficiency is gender dependent. These studies support a role for inflammation in lung cancer susceptibility.

Novel consensus DNA-binding sequence for BRCA1 protein complexes.

Abstract: Increasing evidence continues to emerge supporting the early hypothesis that BRCA1 might be involved in transcriptional processes. BRCA1 physically associates with more than 15 different proteins involved in transcription and is paradoxically involved in both transcriptional activation and repression. However, the underlying mechanism by which BRCA1 affects the gene expression of various genes remains speculative. In this study, we provide evidence that BRCA1 protein complexes interact with specific DNA sequences. We provide data showing that the upstream stimulatory factor 2 (USF2) physically associates with BRCA1 and is a component of this DNA-binding complex. Interestingly, these DNA-binding complexes are downregulated in breast cancer cell lines containing wild-type BRCA1, providing a critical link between modulations of BRCA1 function in sporadic breast cancers that do not involve germline BRCA1 mutations. The functional specificity of BRCA1 tumor suppression for breast and ovarian tissues is supported by our experiments, which demonstrate that BRCA1 DNA-binding complexes are modulated by serum and estrogen. Finally, functional analysis indicates that missense mutations in BRCA1 that lead to subsequent cancer susceptibility may result in improper gene activation. In summary, these findings establish a role for endogenous BRCA1 protein complexes in transcription via a defined DNA-binding sequence and indicate that one function of BRCA1 is to co-regulate the expression of genes involved in various cellular processes.

Gene expression changes following androgen receptor elimination in LNCaP prostate cancer cells.

Abstract: We have shown recently that inhibition of androgen receptor (AR) expression with an antisense AR oligonucleotide (ODN) inhibits LNCaP prostate tumor cells in vitro as well as in vivo. In this study, we investigated gene expression changes that occur after AR signaling blockade, either through AR elimination by antisense treatment or through complete androgen receptor inhibition by androgen deprivation combined with the antiandrogen bicalutamide, in order to search for genes that are directly or indirectly regulated through the AR. Gene expression changes were investigated with cDNA NIH 10K gene microarrays in response to treatment over 48 h. Expression of selected genes was further analyzed by real-time reverse transcriptase (RT)-polymerase chain reaction (PCR), Western blotting, and radioimmunoassay. A comparison of antisense-treated and androgen-deprived cells revealed several concordances such as significant downregulation of prostate-specific genes, cell-cycle regulatory genes, genes of the cholesterol biosynthesis pathway, and several cytoskeletal genes. However, there were also several genes that were differentially regulated. Among the genes that were exclusively changed by treatment with the antisense AR ODN were the insulin-like growth factor binding protein 2 (IGFBP2) and the phosphatidylinositol-4-phosphate 5-kinase type I alpha (PIP5KIA). On the other hand, complete androgen receptor blockade induced changes in the expression of the prostate overexpressed gene 1 and the S100 calcium binding protein P. In summary, we identified a cohort of interesting genes whose expression was highly affected by elimination of the AR in LNCaP prostate cancer cells. Further investigations are warranted to clarify their role in the AR signaling pathway and their susceptibility as a target for the treatment of prostate cancer.

Involvement of c-jun N-terminal kinase activation in 15-deoxy-δ12,14-prostaglandin J2–and prostaglandin A1–induced apoptosis in AGS gastric epithelial cells

Abstract: Cyclopentenone prostaglandins (CyPGs), derivatives of arachidonic acid, have been suggested to exert growth-inhibitory activity through peroxisome proliferator-activated receptor (PPAR)-dependent and -independent mechanisms. Here we examined various eicosanoids for growth inhibition and found that the terminal derivative of prostaglandin (PG) J(2) metabolism, 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)), and PGA(1) markedly inhibited the growth and induced apoptosis in AGS gastric carcinoma cells. There were no significant increases in cell death and DNA-fragmentation in the cells with overexpression of PPARalpha or PPARgamma, indicating the possibility that 15d-PGJ(2) and PGA(1) induced apoptosis through PPAR-independent pathway. Moreover, 15d-PGJ(2) and PGA(1) activated the c-jun N-terminal kinase (JNK) and caspase-3 activity in dose- and time-dependent manners. To examine further the role of JNK signaling cascades in apoptosis induced by 15d-PGJ(2) and PGA(1), we transfected dominant-negative (DN) mutants of JNK plasmid into the cells to analyze the apoptotic characteristics of cells overexpressing DN-JNK following exposure to 15d-PGJ(2) and PGA(1). Overexpression of DN-JNK significantly repressed both endogenous JNK and caspase-3 activity, and subsequently decreased apoptosis induced by 15d-PGJ(2) and PGA(1). These results suggested that CyPGs, such as 15d-PGJ(2) and PGA(1), activated JNK signaling pathway, and that JNK activation may be involved in 15d-PGJ(2)- and PGA(1)-induced apoptosis.

A panel of repeat markers for detection of microsatellite instability in murine tumors.

Abstract: A substantial fraction of human malignancies lack functional DNA mismatch repair (MMR), accumulate mutations at high frequency, and exhibit microsatellite instability (MSI). In order to distinguish between MMR-competent and MMR-deficient malignancies, a consensus panel of microsatellite repeats has been adopted for MSI analysis of human tumors. There is no reference panel of repeats for MSI typing of murine malignancies. In this study, we present six new microsatellite repeat markers for MSI typing of mouse tumors. Analysis of polymerase chain reaction (PCR)-amplified tumor DNA from MMR-deficient and -proficient mice on denaturing polyacrylamide gels revealed that the new panel of microsatellites was more sensitive in detecting MSI than six commonly used CA(n) repeats. Using the new set of microsatellite markers, we demonstrated the presence of MSI in endometrial carcinoma and cancer precursors from diethylstilbestrol (DES)-treated mice, pointing to a possible role for loss of MMR in hormonally promoted endometrial tumorigenesis.