The analysis of mutations and exon deletions at TSC2 gene in angiomyolipomas associated with tuberous sclerosis complex.

Angiomyolipomas (AMLs) are relatively rare hamartomatous or benign tumors that occasionally occur as part of tuberous sclerosis complex (TSC). Mutations in either of the two genes, TSC1 and TSC2, have been attributed to the development of TSC. Between 1994 and January 2009, 83 patients were diagnosed with AML at the Samsung Medical Center. In that group of patients, 5 (6%) had AML with TSC (AML-TSC). Mutational analysis of the TSC2 gene was performed using 7 samples from the 5 AML-TSC patients and 14 samples from 14 patients with sporadic AML without TSC (AML-non-TSC). From this analysis, mutations in TSC genes were identified in 5 samples from the AML-TSC patients (mutation detection rate=71%) and 3 samples from AML-non-TSC patients (mutation detection rate=21%). In the case of AML-TSC, 6 mutations were found including 3 recurrent mutations and 3 novel mutations, while in the case of AML-non-TSC, 4 mutations were identified once, including 1 novel mutation. Also MLPA analysis of the TSC2 gene showed that TSC2 exon deletion is more frequently observed in AML-TSC patients than in AML-non-TSC patients. This is the first mutation and multiplex ligation-dependent probe amplification (MLPA) analyses of TSC2 in Korean AMLs that focus on TSC. This study provides data that are representative of the distribution of mutations and exon deletions at TSC genes in clinically diagnosed AML-TSC cases of the Korean population.

Distinct pattern of allelic loss and inactivation of cadherin 1 and 5 genes in mammary carcinomas arising in p53(+/-) mice.

p53 is one of the most frequently mutated genes in mammary carcinomas (MCs). To detect tumor suppressor genes cooperating with a hetero-deficient p53 gene in mammary carcinogenesis, we first examined allelotypes in MCs from (BALB/cHeA x MSM/Ms) F(1)- p53(+/-) and (BALB/cHeA x 129/SvEv) F(1)- p53(+/-) female mice, and then surveyed down-regulated genes in the allelic loss regions. Genome-wide screening at 42 loci identified frequent (more than 30%) loss of heterozygosity (LOH) on chromosomes 5, 8, 11, 12, 14 and 18 in the MCs from either of the F(1) mice. The MCs in the p53(+/- )mice indicated highly frequent LOH, especially on chromosomes 8, 11 and 12, distinct from other mouse tumors. More than 60% of the 38 MCs from (BALB/cHeA x MSM/Ms) F(1)- p53 (+/-) mice showed LOH in a region ranging from D8Mit85 (105.0 Mb from centromere) to D8Mit113 (111.8 Mb) on chromosome 8, a region syntenic to human chromosome 16q22.1, on which LOH has been found in breast cancers. RT-PCR analyses revealed that the LOH of chromosome 8 was associated with the reduced and/or complete loss of expression of Cdh1 and Cdh5 genes in 15 (58%) and 8 (31%) of 26 MCs derived from the F(1) mice, respectively. Thus, inactivation of Cdh1 and Cdh5 is likely to cooperate with the loss of p53, suggesting a possible tumor suppressive function of these genes in mammary carcinogenesis.

Atm heterozygous deficiency enhances development of mammary carcinomas in p53 heterozygous knockout mice.

INTRODUCTION: Ataxia-telangiectasia is an autosomal-recessive disease that affects neuro-immunological functions, associated with increased susceptibility to malignancy, chromosomal instability and hypersensitivity to ionizing radiation. Although ataxia-telangiectasia mutated (ATM) heterozygous deficiency has been proposed to increase susceptibility to breast cancer, some studies have not found excess risk. In experimental animals, increased susceptibility to breast cancer is not observed in the Atm heterozygous deficient mice (Atm+/-) carrying a knockout null allele. In order to determine the effect of Atm heterozygous deficiency on mammary tumourigenesis, we generated a series of Atm+/- mice on the p53+/- background with a certain predisposition to spontaneous development of mammary carcinomas, and we examined the development of the tumours after X-irradiation. METHODS: BALB/cHeA-p53+/- mice were crossed with MSM/Ms-Atm+/- mice, and females of the F1 progeny ([BALB/cHeA x MSM/Ms]F1) with four genotypes were used in the experiments. The mice were exposed to X-rays (5 Gy; 0.5 Gy/min) at age 5 weeks. RESULTS: We tested the effect of haploinsufficiency of the Atm gene on mammary tumourigenesis after X-irradiation in the p53+/- mice of the BALB/cHeA x MSM/Ms background. The singly heterozygous p53+/- mice subjected to X-irradiation developed mammary carcinomas at around 25 weeks of age, and the final incidence of mammary carcinomas at 39 weeks was 31% (19 out of 61). The introduction of the heterozygous Atm knockout alleles into the background of the p53+/- genotype significantly increased the incidence of mammary carcinoma to 58% (32 out of 55) and increased the average number of mammary carcinomas per mouse. However, introduction of Atm alleles did not change the latency of development of mammary carcinoma. CONCLUSION: Our results indicate a strong enhancement in mammary carcinogenesis by Atm heterozygous deficiency in p53+/- mice. Thus, doubly heterozygous mice represent a useful model system with which to analyze the interaction of heterozygous genotypes for p53, Atm and other genes, and their effects on mammary carcinogenesis.

Generation of large homozygous chromosomal segments by mitotic recombination during lymphomagenesis in F1 hybrid mice.

The loss of heterozygosity (LOH) has been reported in numerous neoplasms in both human and animals, and has often been observed in chromosomal regions, which contain tumor-suppressor genes. We previously found frequent LOH on chromosomes 4, 12 and 19 in radiation-induced lymphomas from (BALB/cHeA x STS/A)F1 hybrid mice by allelotype analysis at polymorphic microsatellite loci. In this study, to elucidate the nature of allelic losses, we refined the loss regions on chromosomes 4, 12 and 19 of the tumors from the F1 mice and then analyzed them cytogenetically. The results represent evidence of a wide range of allelic losses owing to mitotic recombination on chromosomes 4 and 19 in the tumors, possibly reflecting functional losses of putative tumor-suppressor genes. It is suggested that the generation of these large homozygous chromosomal segments probably containing the affected genes is one of the genetic alterations responsible for tumorigenesis.

Preferential induction of mammary tumors in p53 hemizygous BALB/c mice by fractionated irradiation of a sub-lethal dose of X-rays.

BALB/c mice are susceptible to radiation-induced mammary tumors as well as lymphomas. We investigated the effects of the p53 deficient allele and of X-irradiation on the tumor spectrum in the BALB/c background. Substantially all p53 -/-animals died of thymic lymphomas before 36 weeks of age, while none of the p53 +/+ animals died during that period. At this age, mortalities of p53 +/- females and males were 5% (1/22) and 11% (1/9), respectively, due to non-thymic lymphoma and sarcoma. When exposed to 4 Gy of X-irradiation, 100% (44/44) and 95% (18/19) of p53 +/- mice died with tumors within 36 weeks. Among these, the predominant cause of death was lymphoma in either sex [26/44 (59%) in females; 13/19 (68%) in males]; mammary adenocarcinoma (15/44, 34%) and sarcoma (3/19, 16%) were semi-dominant in females and males, respectively. The mortalities of similarly treated p53 +/+ mice were 16% (5/31) in females and 17% (3/18) in males: virtually all deaths were due to thymic lymphomas in either sex. When exposed to 4 currency 0.7 Gy of X-irradiation at weekly intervals, 23/23 (100%) of the p53 +/-females died of tumors within 36 weeks. In these animals, mammary adenocarcinoma (15/23, 65%), instead of lymphoma (7/23, 30%), was dominant. None of the similarly treated p53 +/+ females developed malignant tumors during the period. Mammary adenocarcinomas generated in p53 +/- females exposed or non-exposed to radiation showed a frequent loss of the p53 wild-type allele. Hence, we provided a useful experimental system to study radiation-induced mammary tumors in mice.

Putative tumor-suppressor gene regions responsible for radiation lymphomagenesis in F1 mice with different p53 status.

Regions of allelic loss on chromosomes in many tumors of human and some experimental animals are generally considered to harbor tumor-suppressor genes involved in tumorigenesis. Allelotype analyses have greatly improved our understanding of the molecular mechanism of radiation lymphomagenesis. Previously, we and others found frequent loss of heterozygosity (LOH) on chromosomes 4, 11, 12, 16 and 19 in radiation-induced lymphomas from several F1 hybrid mice. To examine possible contributions of individual tumor-suppressor genes to tumorigenesis in p53 heterozygous deficiency, we investigated the genome-wide distribution and status of LOH in radiation-induced lymphomas from F1 mice with different p53 status. In this study, we found frequent LOH (more than 20%) on chromosomes 4 and 12 and on chromosomes 11, 12, 16 and 19 in radiation-induced lymphomas from (STS/A X MSM/Ms)F1 mice and (STS/A X MSM/Ms)F1-p53KO/+ mice, respectively. Low incidences of LOH (10-20%) were also observed on chromosomes 11 in mice with wild-type p53, and chromosomes 1, 2, 9, 17 and X in p53 heterozygous-deficient mice. The frequency of LOH on chromosomes 9 and 11 increased in the (STS/A X MSM/Ms)F1-p53KO/+ mice. Preferential losses of the STS-derived allele on chromosome 9 and wild-type p53 allele on chromosome 11 were also found in the p53 heterozygous-deficient mice. Thus, the putative tumor-suppressor gene regions responsible for lymphomaganesis might considerably differ due to the p53 status.

Allelic loss analysis of lymphomas induced in Fas-heterozygous deficient mice.

Mutations of Fas (CD95/Apo-1) gene have been reported in various malignancies and therefore the Fas gene has been considered to be a tumor suppressor gene. To examine an involvement of Fas gene as a tumor suppressor gene in radiation lymphomagenesis, we examined the loss of heterozygosity (LOH) in lymphomas from (MSM/Ms x MRL-MpJ/Fas (lpr)) F(1) and (BALB/cHeA x MRL-MpJ/Fas (lpr)) F(1) hybrid mice. Lymphoma development by X-irradiation was efficiently observed in both F(1) hybrids. Frequent LOH was found on chromosomes 12 and 4 in the tumors from both F(1) mice, but no allelic loss on chromosome 19 containing Fas locus was found, and no wild-type allele of the Fas gene was lost in 51 lymphomas. Therefore, the putative tumor-suppressor gene regions responsible for lymphomagenesis might not considerably differ due to the Fas gene status.

Human telomerase reverse transcriptase is a promising target for cancer inhibition in squamous cell carcinomas.

BACKGROUND/AIM: The present study aimed to investigate whether the down-regulation of human telomerase reverse transcriptase (hTERT) may induce an anti-invasive effect in oral squamous cell cancer cell lines. MATERIALS AND METHODS: A genetically-engineered squamous carcinoma cell line overexpressing hTERT in immortalized oral keratinocytes transfected by human papilloma virus (HPV)-16 E6/E7 (IHOK) was used. In vivo tumorigenicity was examined using an orthotopic xenograft model of nude mice. For evaluating anti-invasive activity by knockdown of hTERT expression, transwell invasion assay and real-time polymerase chain reaction (PCR) for matrix metalloproteinases (MMP) were employed. RESULTS: The down-regulation of hTERT expression reduced the invasive activity and MMP expression. This result was re-confirmed in the HSC3 oral squamous carcinoma cell line. CONCLUSION: Targeting hTERT may lead to novel therapeutic approaches.

Gankyrin is a predictive and oncogenic factor in well-differentiated and dedifferentiated liposarcoma.

Liposarcoma is one of the most common histologic types of soft tissue sarcoma and is frequently an aggressive cancer with poor outcome. Hence, alternative approaches other than surgical excision are necessary to improve treatment of well-differentiated/dedifferentiated liposarcoma (WDLPS/DDLPS). For this reason, we performed a two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry/mass spectrometry (MALDI-TOF/MS) analysis to identify new factors for WDLPS and DDLPS. Among the selected candidate proteins, gankyrin, known to be an oncoprotein, showed a significantly high level of expression pattern and inversely low expression of p53/p21 in WDLPS and DDLPS tissues, suggesting possible utility as a new predictive factor. Moreover, inhibition of gankyrin not only led to reduction of in vitro cell growth ability including cell proliferation, colony-formation, and migration, but also in vivo DDLPS cell tumorigenesis, perhaps via downregulation of the p53 tumor suppressor gene and its p21 target and also reduction of AKT/mTOR signal activation. This study identifies gankyrin, for the first time, as new potential predictive and oncogenic factor of WDLPS and DDLPS, suggesting the potential for service as a future LPS therapeutic approach.

Dryopteris crassirhizoma has anti-cancer effects through both extrinsic and intrinsic apoptotic pathways and G0/G1 phase arrest in human prostate cancer cells.

AIM OF THE STUDY: The inhibitory effect of Dryopteris crassirhizoma on the proliferation of human metastatic prostate PC3-MM2 cells and the mechanism of action were examined to identify its anti-cancer properties. The effect of the extract on cell cycle progression and its combined cytotoxic effect with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on PC3-MM2 cells were also investigated. MATERIALS AND METHODS: The anti-proliferative effects of Dryopteris crassirhizoma were examined by culturing PC3-MM2 cells in the presence or absence of various concentrations of Dryopteris crassirhizoma extract, and the inhibitory effects on cell proliferation were determined by Cell Counting Kit (CCK)-8 analysis. The quantities of apoptosis-inducing proteins were measured by western blotting analysis. Cell cycle progression was analyzed by PI staining using flow cytometry. RESULTS: Dryopteris crassirhizoma (50 and 100 microg/ml) inhibited markedly the proliferation of PC-3 and PC3-MM2 cells without cytotoxicity to normal (spleen) cells from BALB/C mice. Dryopteris crassirhizoma (100 microg/ml) effectively induced apoptosis through the activation of caspase-3, -8, -9, bid, and PARP in PC3-MM2 cells. The cells exposed to Dryopteris crassirhizoma increased significantly the accumulation of the DNA contents in the G0/G1 phase and sub-G1 phase in contrast to the control. The combined cytotoxic effects of Dryopteris crassirhizoma and TRAIL induced the increased activity of 29% in contrast to the sum of the inhibitory effects of each agent alone. CONCLUSIONS: Dryopteris crassirhizoma has anti-cancer properties by inducing cell cycle arrest and apoptosis through the extrinsic and intrinsic pathway in PC3-MM2 cells. The extract also showed a combined effect with TRAIL on the inhibition of proliferation in the cells. These findings suggest that possibly its extract could be used for treating androgen-independent prostate cancer with minimal side effects.

Differential gene expression profiles in the steatosis/fibrosis model of rat liver by chronic administration of carbon tetrachloride.

Global gene expression profile was analyzed by microarray analysis of rat liver RNA after chronic carbon tetrachloride (CCl(4)) administration. Rats received 0.5 ml CCl(4)/kg three times a week, and the liver samples were obtained after 0, 30, 60, and 90 days of injection. Histopathologic studies of liver tissues enabled the classification of the CCl(4) effect into mild and severe fatty liver/steatosis (30 and 60 days, respectively) and fibrosis/cirrhosis (90 days) stages. The expression levels of 4,900 clones on a custom rat gene microarray were analyzed and the results were confirmed by semi-quantitative RT-PCR. Four hundred thirty-eight clones were differentially expressed with more than a 1.625-fold difference (which equals 0.7 in log2 scale) at one or more time points. Multiple genes involved in lipid metabolism and ribosome biogenesis showed differential transcript levels upon chronic CCl(4) administration, which was previously seen in acute rat model as well. In addition, a total of 149 clones were identified as fibrosis/cirrhosis-specific genes by either fold changes or Significance Analysis of Microarrays. In conclusion, we report microarray analysis results in rat liver upon chronic CCl(4) administration with a full chronological profile that not only covered fatty liver/steatosis but also later points of fibrosis/cirrhosis. These data will provide the insight of specific gene expression profiles that is implicated in the multistep process of fatty liver/steatosis and fibrosis/cirrhosis after chronic hepatotoxin exposure.

Comprehensive analysis of differential gene expression profiles on carbon tetrachloride-induced rat liver injury and regeneration.

Microarray analysis of RNA from carbon tetrachloride (CCl4)-administered rat livers was performed at various time points to establish a global gene expression profile during injury and regeneration stages. A single dose of 1 ml/kg of CCl4 was given by ip injection, and the liver samples were obtained after 6, 24, 48 h, and 2 weeks. Histopathologic, biochemical, and immunohistochemical studies enabled the classification of the CCl4 effect into injury (6 and 24 h) and regeneration (48 h and 2 weeks) stages. The expression levels of 5180 clones on a custom rat gene microarray were analyzed and 587 clones yielded changeable gene expression on at least single time point. One hundred seventy-nine clones were classified as injury-specific clones, while 38 clones as regeneration-specific clones. Characteristic gene expression profiles could be associated with CCl4-induced gene expression with the disruption of lipid metabolism, which is known to cause the fatty liver induced by CCl4 treatment. In addition, induction of the transcripts for many ribosomal proteins was detected during the injury stage, particularly at the 24-h time point, despite the previous report of decreased protein synthesis rate upon CCl4 treatment. Several genes with known functions were also identified as CCl4-regulated genes. In conclusion, we established a global gene expression profile utilizing microarray analysis in rat liver upon acute CCl4 administration with a full chronological profile that not only covers injury stage but also later points of regeneration stage.