Overexpression of Replication-Dependent Histone Signifies a Subset of Dedifferentiated Liposarcoma with Increased Aggressiveness.

Liposarcoma (LPS) is an adult soft tissue malignancy that arises from fat tissue, where well-differentiated (WD) and dedifferentiated (DD) forms are the most common. DDLPS represents the progression of WDLPS into a more aggressive high-grade and metastatic form. Although a few DNA copy-number amplifications are known to be specifically found in WD- or DDLPS, systematic genetic differences that signify subtype determination between WDLPS and DDLPS remain unclear. Here, we profiled the genome and transcriptome of 38 LPS tumors to uncover the genetic signatures of subtype differences. Replication-dependent histone (RD-HIST) mRNAs were highly elevated and their regulation was disrupted in a subset of DDLPS, increasing cellular histone molecule levels, as measured using RNA-seq (the averaged fold change of 53 RD-HIST genes between the DD and WD samples was 10.9) and immunohistochemistry. The change was not observed in normal tissues. Integrated whole-exome sequencing, RNA-seq, and methylation analyses revealed that the overexpressed HMGA2 (the fold change between DD and WD samples was 7.3) was responsible for the increased RD-HIST level, leading to aberrant cell proliferation. Therefore, HMGA2-mediated elevation of RD-HISTs were crucial events in determining the aggressiveness of DDLPS, which may serve as a biomarker for prognosis prediction for liposarcoma patients.

Complete regression of metastatic de-differentiated liposarcoma with engineered mesenchymal stromal cells with dTRAIL and HSV-TK.

De-differentiated liposarcoma (DDLPS) is a rare cancer with high rates of recurrence and metastasis. Currently, treatment with doxorubicin-ifosphamide, following surgical resection, is routinely performed. However, clinical treatment of these refractory cancers require further study. We investigated the treatment of mesenchymal stromal cells (MSC) transduced with dodecameric tumor necrosis factor receptor apoptosis-inducing ligand (dTRAIL) and herpes simplex virus thymidine kinase (HSV-TK) (MSC-TR/TK), as a method to approach DDLPS therapy. First, in order to assess the efficacy of this therapy, cell viability was evaluated by apoptosis analysis of a DDLPS cell line co-cultured with patient-derived cells (PDCs) and MSC-TR/TK in vitro. In vivo, we established a lung metastasis model using the DDLPS cell line and assessed the anti-tumorigenic efficiency of dTRAIL-TK by injecting MSC-TR/TK. Results confirmed that liposarcoma cells resistant to dTRAIL in PDCs, transformed by HSV-TK, induced apoptosis effectively after treatment with toxic ganciclovir (GCV). Meanwhile, we observed that treatment of GCV after injection of MSC-TR/TK effectively eliminated lung nodules in a lung metastasis model established from LPS246 cells resistant to dTRAIL. When mice were treated with GCV two days after double injection with MSC-TR/TK, the tumor suppression effect was even more pronounced.

Anti-tumor effects of anti-PD-1 antibody, pembrolizumab, in humanized NSG PDX mice xenografted with dedifferentiated liposarcoma.

The efficacy of an immune checkpoint blockade has been demonstrated against various types of cancer, but its suitability has not been fully proven for therapies specifically targeting sarcoma. We conducted a pan-cancer tumor data analysis to identify key immune-related variables strongly associated with sarcoma prognosis, and we explored whether these expected factors are functionally correlated with anti-PD-1 therapy in humanized (Hu) NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice xenografted with dedifferentiated liposarcoma (DDLPS). We found that an abundance of hCD8+ T cells and hNK cells was functionally associated with anti-PD-1 effects in the Hu-NSG DDLPS mice. Phenotypically, these cells were shown to be hCD8+IFNγ+, hCD8+PD-1+, hCD8+Ki-67+, hCD56+IFNγ+, hCD56+PD-1+, and hCD56+Ki-67+ cells and were enriched in splenocytes and tumor-infiltrating lymphocytes (TILs) of Hu-NSG DDLPS mice treated with anti-PD-1 antibody. Moreover, a considerable increase in activated hCD56+NKp46+NKG2D+ NK cells was also detected. Our findings suggest that hCD8+ T and hNK subsets play a pivotal role in anti-DDLPS tumor effects of anti-PD-1 therapy. The results provide clinical reference for advanced anti-PD-1 therapy targeting sarcoma tumors including DDLPS.

Combination therapy with c-met inhibitor and TRAIL enhances apoptosis in dedifferentiated liposarcoma patient-derived cells.

BACKGROUND: Liposarcoma (LPS) is a tumor derived from adipose tissue, and has the highest incidence among soft tissue sarcomas. Dedifferentiated liposarcoma (DDLPS) is a malignant tumor with poor prognosis. Recurrence and metastasis rates in LPS remain high even after chemotherapy and radiotherapy following complete resection. Therefore, the development of advanced treatment strategies for LPS is required. In the present study, we investigated the effect of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment, and of combination treatment using TRAIL and a c-Met inhibitor on cell viability and apoptosis in LPS and DDLPS cell lines of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment, and of combination treatment using TRAIL and a c-Met inhibitor. METHODS: We analyzed cell viability after treatment with TRAIL and a c-Met inhibitor by measuring CCK8 and death receptor 5 (DR5) expression levels via fluorescence activated cell sorting (FACS) in both sarcoma cell lines and DDLPS patient-derived cells (PDCs). Moreover, we validated the effects of TRAIL alone and in combination with c-Met inhibitor on apoptosis in LPS cell lines and DDLPS PDCs via FACS. RESULTS: Our results revealed that combination treatment with a c-Met inhibitor and human recombinant TRAIL (rhTRAIL) suppressed cell viability and induced cell death in both sarcoma cell lines and DDLPS PDCs, which showed varying sensitivities to rhTRAIL alone. Also, we confirmed that treatment with a c-Met inhibitor upregulated DR5 levels in sarcoma cell lines and DDLPS PDCs. In both TRAIL-susceptible and TRAIL-resistant cells subjected to combination treatment, promotion of apoptosis was dependent on DR5 upregulation. CONCLUSION: From these results, our findings validated that DR5 up-regulation caused by combination therapy with a c-Met inhibitor and rhTRAIL enhanced TRAIL sensitization and promoted apoptosis. We propose the use of this approach to overcome TRAIL resistance and serve as a novel treatment strategy for clinical trials.

Establishment of a Novel PDX Mouse Model and Evaluation of the Tumor Suppression Efficacy of Bortezomib Against Liposarcoma.

The patient-derived xenograft (PDX) model has been adopted as a major tool for studying tumorigenesis and differentiation in various carcinomas. In addition, it has been used in the development of anticancer agents. PDX models have been among the most meaningful tools used to understand the role of stromal cells and vascular cells in the body, which are major factors in cancer development and the application of therapeutic agents. Also, the establishment of PDX models from liposarcoma patients is considered to be important for understanding lipomagenesis and following drugs development. For these reasons, we developed patient-derived cell (PDC) and PDX models derived from 20 liposarcoma patients. The tissues of these patients were obtained in accordance with the principles of the Samsung Medical Center's ethics policy, and cell culture and xenografting onto the mice were performed under these principles. High-throughput drug screening (HTS) was carried out using established PDCs to select candidate drugs. Among the different candidate anticancer drugs, we tested the effect of bortezomib, which was expected to inhibit MDM2 amplification. First, we confirmed that the PDCs maintained the characteristics of liposarcoma cells by assessing MDM2 amplification and CDK4 overexpression using fluorescence in situ hybridization. Analysis of short tandem repeats and an array using comparative genomic hybridization confirmed that the PDX model exhibited the same genomic profile as that of the patient. Immunohistochemistry for MDM2 and CDK4 showed that the overexpression patterns of both proteins were similar in the PDX models and the PDCs. Specifically, MDM2 amplification was observed to be significantly correlated with the successful establishment of PDX mouse models. However, CDK4 expression did not show such a correlation. Of the anticancer drugs selected through HTS, bortezomib showed a strong anticancer effect against PDC. In addition, we observed that bortezomib suppressed MDM2 expression in a dose-dependent manner. In contrast, p21 tended to elicit an increase in PDC expression. Treatment of the PDX model with bortezomib resulted in an anticancer effect similar to that seen in the PDCs. These results support that PDCs and PDX models are among the most powerful tools for the development and clinical application of anticancer drugs for the treatment of liposarcoma patients.

Tumor Suppression Efficacy of Heat Shock Protein 90 Inhibitor 17AAG in a Liposarcoma Mouse Model.

BACKGROUND/AIM: Heat shock protein 90 (HSP90) inhibitors have recently been tested as anticancer drugs in a variety of carcinomas. Yet, there exist only few reports about HSP90 inhibitor and its thepeutic effect on liposarcoma. The therapeutic effects of HSP90 inhibitors have been mainly observed in oncogenic and tumor angiogenic signaling cascades by observing tumor growth. MATERIALS AND METHODS: We used the the LPS 246 liposarcoma cell line and GS-076 PDC (patient-derived cell lines). On these, we performed cell viability assays and migration assays under treatment with the HSP90 inhibitor, 17AAG. For analyzing angiogenesis factor, we used quantitative polymerase chain reaction (qPCR) after treating cells with the 17AAG inhibitor. Regarding in vivo assay, we made the tumor model in immune-deficient mouse and compared the tumor size of drug-treated group at each time point with controls. For sequestering analysis of angiogenesis factor in vivo, we performed immuno-fluorescence (IF) staining on tumor tissue. RESULTS: Through cell viability, migration assay and qPCR about angiogenesis factor, we demonstrated the anti-oncogenic and anti-angiogenic effects of an HSP90 inhibitor on a liposarcoma cell line and a patient-derived primary cell model (PDC). Also, the HSP90 inhibitor 17AAG effectively inhibited the activity of protein kinase B (AKT) and blocked extracellular signal-regulated kinase (ERK) activity. Hence, 17AAG effectively disrupted the oncogenic signaling cascade and substantially inhibited tumor growth in vitro. In an LPS 863 cell xenograft mouse model treated with 17AAG, we observed that tumor size was decreasing, as well as down-regulation of the expression levels of vascular endothelial growth factor receptor 2 (VEGFR2), CD31 and signal transducer and activator of transcription-3 (STAT3). CONCLUSION: 17AAG reduced the activity of AKT, ERK, VEGF and STAT3 in oncogenic and angiogenic pathways in liposarcoma PDC models derived from patients' tissues and cancer cell lines.

Therapeutic strategies for locally recurrent and metastatic de-differentiated liposarcoma with herpes simplex virus-thymidine kinase-expressing mesenchymal stromal cells.

BACKGROUND AIMS: Major challenges in de-differentiated liposarcoma (DDLPS) therapy are the high rate of sequential recurrence (>80%) and metastasis (20-30%) following surgical removal. However, well-defined therapeutic strategies for this rare malignancy are lacking and are critically needed. METHODS: We investigated a new approach to DDLPS therapy with mesenchymal stromal cells expressing herpes simplex virus-thymidine kinase (MSC-TK). In an effort to evaluate this efficacy, in vitro cytotoxicity of MSC-TK against DDLPS cells was analyzed using an apoptosis assay. For pre-clinical study, the MSC-TK-induced reduction in recurrence and metastasis was validated in a recurrent DDLPS model after the macroscopic complete resection and lung metastasis DDLPS model. RESULTS: MSC-TK induced apoptosis in DDLPS cells by bystander effects via gap junction intracellular communication (GJIC) of toxic ganciclovir (GCV). Recurrent DDLPS models following no residual tumor/microscopic tumor resection and lung metastasis DDLPS models were established, which suggested clinical relevance. MSC-TK markedly reduced locoregional recurrence rates and prolonged recurrence-free survival, thus increasing overall survival in the recurrent DDLPS model. MSC-TK followed by GCV treatment yielded a statistically significant reduction in early- and advanced-stage lung metastasis. DISCUSSION: This therapeutic strategy may serve as an alternative or additional strategy by applying MSC-TK to target residual tumors following surgical resection, thus reducing local relapse and metastasis in these patients.

SIRT1 regulates oncogenesis via a mutant p53-dependent pathway in hepatocellular carcinoma.

BACKGROUND & AIMS: SIRT1 is a class III histone deacetylase that plays diverse roles in various cancers. However, the clinical significance of SIRT1 in hepatocellular carcinoma (HCC) remains unknown. METHODS: We analysed p53 mutations and the activation of SIRT1 in 252 hepatitis B virus-positive HCC cases. None of the patients had been subjected to pre-operative treatment. RESULTS: We examined 57 p53 mutations from 248 HCC tissues. Activated SIRT1 (phosphorylated form of Ser47), in the context of mutant p53, predicted a longer relapse-free survival (RFS) but not a longer overall survival (OS) (RFS: p = 0.007, OS: p = 0.280) in HCC tissues harbouring mutant p53. In multivariate analysis, activated SIRT1 remained a significant predictor of longer RFS (OR = 0.307, CI: 0.143-0.660, p=0.002). Analysis of 248 paired specimens revealed a significant correlation between activated SIRT1 (Ser47) and activated AMPK (Thr172) in HCC tissues harbouring mutant p53 (p = 0.003, n = 57). The combination of these 2 parameters was a powerful predictor for a good prognosis in these patients. In vitro, SIRT1 inactivation stimulated the growth of HCC cells, bearing mutated p53, by suppressing AMPK activity and subsequently enhancing mammalian target of rapamycin (mTOR) activity, resulting in induction of p70S6K1 activation in HCC cells. Metformin, an AMPK activator, more strongly suppressed cell growth in p53-mutant cell lines with inactive SIRT1 than in p53-mutant cell lines with active SIRT1. CONCLUSIONS: SIRT1 exerted anti-carcinogenic effects via the AMPK-mTOR pathway in HCC in the context of mutant p53. Metformin could be a therapeutic drug for HCC in patients with mutated p53, inactivated SIRT1, and AMPK expression.

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.

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.

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.

Expression and functional significance of the erythropoietin receptor in hepatocellular carcinoma.

BACKGROUND: Erythropoietin, through its specific receptor (EpoR), may induce responses in a variety of non-haematopoietic tissues including malignant cells. The purpose of this study was to examine the expression of EpoR in hepatocellular carcinoma (HCC) and to correlate the levels of EpoR expression with the clinicopathological properties of HCC and tumour recurrence. METHODS: The study included 134 patients who underwent curative hepatectomy for hepatitis B virus (HBV)-related primary HCC. The clinical, laboratory and pathological data from these patients were retrospectively collected. The expression of EpoR mRNA and protein were evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. RESULTS: Expression of EpoR mRNA in the cirrhotic liver was positively correlated with tumour cell differentiation and 1-year disease-free survival (74.8% in the high expression group versus 46.9% in the low expression group; P = 0.001), as it was for EpoR mRNA expression in HCC (64.4% in the high expression group versus 52.7% in the low expression group; P = 0.044). Tumour recurrence showed stronger dependence on the expression of EpoR protein in non-malignant cirrhotic livers than in HCC. CONCLUSION: In HBV-related HCC, the levels of EpoR mRNA and protein in non-tumour cirrhotic livers were positively correlated with tumour cell differentiation, which is a favourable predictor of disease-specific survival.

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.

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.

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.

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.

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.

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.