Primary Tumor Cell Cultures Research Articles

Synthesis and Selective Anticancer Activity of Organochalcogen Based Redox Catalysts

Many tumor cells exhibit a disturbed intracellular redox state resulting in higher levels of reactive oxygen species (ROS). As these contribute to tumor initiation and sustenance, catalytic redox agents combining significant activity with substrate specificity promise high activity and selectivity against oxidatively stressed malignant cells. We describe here the design and synthesis of novel organochalcogen based redox sensor/effector catalysts. Their selective anticancer activity at submicromolar and low micromolar concentrations was established here in a range of tumor entities in various biological systems including cell lines, primary tumor cell cultures, and animal models. In the B-cell derived chronic lymphocytic leukemia (CLL), for instance, such compounds preferentially induce apoptosis in the cancer cells while peripheral blood mononuclear cells (PBMC) from healthy donors and the subset of normal B-cells remain largely unaffected. In support of the concept of sensor/effector based ROS amplification, we are able to demonstrate that underlying this selective activity against CLL cells are pre-existing elevated ROS levels in the leukemic cells compared to their nonmalignant counterparts. Furthermore, the catalysts act in concert with certain chemotherapeutic drugs in several carcinoma cell lines to decrease cell proliferation while showing no such interactions in normal cells. Overall, the high efficacy and selectivity of (redox) catalytic sensor/effector compounds warrant further, extensive testing toward transfer into the clinical arena.

A combined strategy of serial analysis of gene expression (SAGE) and quantitative PCR (qPCR) to identify four genes that predict outcome in non-small cell lung cancer patients.

e17502 Background: Cisplatin-based adjuvant chemotherapy improves survival among patients with completely resected non-small cell lung cancer. However, objective response rates are in the range of 30% to 50%. Thus, prediction of treatment response and outcome before CRT is a major challenge for clinicians. Methods: In a first stage we perfomed a SAGE analysis on snap-frozen surgical specimens from three good responder and three bad responder patients. Response sensibility to cisplatin was evaluated using cytotoxicity assays in primary cultures of tumor cells from the surgical specimens. In a second stage we analyzed the expression of 22 genes and 2 controls using TaqMan low density arrays in snap-frozen surgical specimens from 51 operable stages non-small cell lung cancer patients. Disease-free survival was used as the endpoint of the study. All patients received cisplatin-based adjuvant chemotherapy. Multivariate Cox methods were applied to build an optimized predictor of outcome. Results: The combination of mTOR, TWIST1, AKCR1C2, and HSPB1 gene expression was able to predict clinical outcome in pretreated tissues. In addition, DFS analysis revealed that the gene signature- based score was the only statistically significant prognostic factor remaining in the multivariate analysis. Conclusions: A basal 4-gene signature, obtained from surgical specimens of non-small cell lung cancer, efficiently predicted clinical outcome in the patients. These preliminary results are very encouraging and, if they are validated in larger series, could have clinical implications. No significant financial relationships to disclose.

Abstract 4242: TRP53 DELETION INDUCES PROLONGED SURVIVAL OF BIPOTENT LIVER STEM CELLS AND THE EVOLUTION OF BI-DIFFERENTIATED HCC/CC

Abstract Background: Defects in the p53-signaling checkpoint have been described in 30-60% of human hepatocellular carcinoma (HCC) and are associated with less differentiated tumors and poor prognosis. The contribution of p53 deletion by itself to hepatocarcinogenesis is not known. Furthermore it is not clear, if liver stem cells play a role in the onset of liver tumor formation. Methods: We generated mice with liver specific TRP53 deletion (TRP53−/−) by crossing conditional TRP53 KO mice with AlfpCRE transgenic mice (CRE expression under the albumin-alphafetoprotein-enhancer-promoter). In vitro cultures were established from the developing liver tumors and analyzed for potential tumor stem cells using xenotransplantation. Endogenous liver stem/progenitor cells were analysed using FACS purification and in vitro culturing of purified liver stem cells (CD13+CD133+CD49f+). Results: Mice with liver specific TRP53 deletion developed liver tumors with a mixed differentiation (hepatocellular and cholangiocellular carcinoma). Purification and xenotransplantation of subpopulations of tumor cells from primary tumor cell cultures (using following surface marker: CD13, CD90, CD133, or cKit) revealed no differences in tumor forming capacity in NMRI nu/nu mice. Moreover, tumor formation was seen after xenotransplantation of very small tumor cell numbers (2-cells) indicating that the tumors have an inherent high tumor forming capacity. An analysis of the colony formation capacity of endogenous liver stem/progenitor cells revealed a prolonged survival of bipotent liver stem cells in aged TRP53−/− mice compared to TRP53+/− or TRP53+/+ mice. The last two groups showed a complete loss of bipotent liver stem cells by 8 month of age, whereas bipotent liver stem cells were detected with a frequency of 0,4% in liver of 8-10 month old TRP53−/− mice. Conclusion: This study provides the first experimental evidence that TRP53 deletion leads to a prolonged survival of bipotent liver stem cells and the formation of bi-differentiated liver tumors. These data indicate that the p53-dependent tumor suppressor functions include the restriction of proliferation and survival of bipotent stem cells in the liver. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4242.

Microenvironmental modulation of asymmetric cell division in human lung cancer cells

Normal tissue homeostasis is maintained through asymmetric cell divisions that produce daughter cells with differing self-renewal and differentiation potentials. Certain tumor cell subfractions can self-renew and repopulate the heterogeneous tumor bulk, suggestive of asymmetric cell division, but an equally plausible explanation is that daughter cells of a symmetric division subsequently adopt differing cell fates. Cosegregation of template DNA during mitosis is one mechanism by which cellular components are segregated asymmetrically during cell division in fibroblast, muscle, mammary, intestinal, and neural cells. Asymmetric cell division of template DNA in tumor cells has remained elusive, however. Through pulse-chase experiments with halogenated thymidine analogs, we determined that a small population of cells within human lung cancer cell lines and primary tumor cell cultures asymmetrically divided their template DNA, which could be visualized in single cells and in real time. Template DNA cosegregation was enhanced by cell-cell contact. Its frequency was density-dependent and modulated by environmental changes, including serum deprivation and hypoxia. In addition, we found that isolated CD133(+) lung cancer cells were capable of tumor cell repopulation. Strikingly, during cell division, CD133 cosegregated with the template DNA, whereas the differentiation markers prosurfactant protein-C and pan-cytokeratins were passed to the opposing daughter cell, demonstrating that segregation of template DNA correlates with lung cancer cell fate. Our results demonstrate that human lung tumor cell fate decisions may be regulated during the cell division process. The characterization and modulation of asymmetric cell division in lung cancer can provide insight into tumor initiation, growth, and maintenance.

Altered Expression of Neurotensin Receptors Is Associated with the Differentiation State of Prostate Cancer

In prostate cancer, traditional treatments such as androgen response manipulation often provide only temporary resolution of disease, with emergence of a more aggressive, androgen-independent tumor following initial therapy. To treat recurrent disease, cell surface proteins that are specifically overexpressed on malignant cells may be useful for generating targeted therapeutics. Recent evidence suggests that neurotensin receptors (NTR) are recruited in advanced prostate cancer as an alternative growth pathway in the absence of androgens. In this study, we assessed the potential use of these receptors as targets by analyzing NTR expression patterns in human prostate cell lines and primary prostate tumor cell cultures derived from patient samples. In primary tumor cell cultures, NTR1 was upregulated in cells with a basal phenotype (cytokeratin 1/5/10/14+), whereas NTR2 and NTR3 were upregulated in cells with luminal phenotype (cytokeratin 18+). Similar patterns of NTR expression occurred in benign prostate tissue sections, implicating differentiation state as a basis for the differences observed in tumor cell lines. Our findings support the use of NTRs as tools for therapeutic targeting in prostate cancers composed of both poorly differentiated and/or well-differentiated cells.

Characterization of the cytotoxic properties of the benzimidazole fungicides, benomyl and carbendazim, in human tumour cell lines and primary cultures of patient tumour cells

The benzimidazoles, benomyl and carbendazim, are fungicides suggested to target microtubules. Benomyl is metabolized to carbendazim, which has already been explored as an anticancer drug in phase 1 clinical trials. We further characterized the cytotoxic properties of benomyl and carbendazim in 12 human cell lines and in primary cultures of patient tumour cells with the overall aims of elucidating mechanisms of action and anticancer activity spectrum. Cytotoxicity was assessed in the short-term fluorometric microculture cytotoxicity assay and was correlated with the activity of other anticancer drugs and gene expression assessed by cDNA microarray analysis. Benomyl was generally more potent than its metabolite, carbendazim. Both showed high drug activity correlations with several established and experimental anticancer drugs, but modest association with established mechanisms of drug resistance. Furthermore, these benzimidazoles showed high correlations with genes considered relevant for the activity of several mechanistically different standard and experimental anticancer drugs, indicating multiple and broad mechanisms of action. In patient tumour samples, benomyl tended to be more active in haematological compared with solid tumour malignancies, whereas the opposite was observed for carbendazim. In conclusion, benomyl and carbendazim show interesting and diverse cytotoxic mechanisms of action and seem suitable as lead compounds for the development of new anticancer drugs.

Evaluation of toxicity and antitumor activity of cycloviolacin O2 in mice

Cycloviolacin O2 is a small cyclic cysteine-rich protein belonging to the group of plant proteins called cyclotides. This cyclotide has been previously shown to exert cytotoxic activity against a variety of human tumor cell lines as well as primary cultures of human tumor cells in vitro. This study is the first evaluation of its tolerability and antitumor activity in vivo. Maximal-tolerated doses were estimated to 1.5 mg/kg for single intravenous (i.v.) dosing and 0.5 mg/kg for daily repeated dosing, respectively. Two different in vivo methods were used: the hollow fiber method with single dosing (i.v., 1.0 mg/kg) and traditional xenografts with repeated dosing over 2 weeks (i.v., 0.5 mg/kg daily, 5 days a week). The human tumor cell lines used displayed dose-dependent in vitro sensitivity (including growth in hollow fibers to confirm passage of cycloviolacin O2 through the polyvinylidene fluoride fibers), with IC5o values in the micromolar range. Despite this sensitivity in vitro, no significant antitumor effects were detected in vivo, neither with single dosing in the hollow fiber method nor with repeated dosing in xenografts. In summary, the results indicate that antitumor effects are minor or absent at tolerable (sublethal) doses, and cycloviolacin O2 has a very abrupt in vivo toxicity profile, with lethality after single injection at 2 mg/kg, but no signs of discomfort to the animals at 1.5 mg/kg. Repeated dosing of 1 mg/kg gave a local-inflammatory reaction at the site of injection after 2-3 days; lower doses were without complications.

Expression of cyclooxygenase-2 (COX-2) in an advanced metastasized hypopharyngeal carcinoma and cultured tumor cells

The inducible enzyme cyclooxygenase-2 (COX-2) catalyzes PGE(2) production and plays an important role in the progression of many solid cancers. However, the role of COX-2 expression in cervical lymph node metastases of head and neck cancer has not been clarified yet. We comment on a male patient aged 53 who was admitted to an ENT-department with acute bleeding from an advanced hypopharyngeal carcinoma and a frontotemporal mass. Prior to palliative intended radiotherapy, the metastasis was resected. During the procedure, a small amount of tumor tissue was harvested for primary tumor cell culture. COX-2 overexpression was demonstrated in the primary tumor tissue, the metastasis, in the cultured tumor cells by standard immunohistochemistry, as well as cytochemistry. A simultaneous expression of COX-2 in head and neck carcinoma was presented for the first time. Besides the prognostic impact in oral carcinogenesis, this COX-2 role of biomarker for aggressive head and neck squamous cell carcinomas should be further evaluated. Additionally, treatment of hypopharyngeal carcinomas with selective COX-2 inhibitors could be beneficial when administered in combination with radiochemotherapy.

Coassociation of Estrogen Receptor and p160 Proteins Predicts Resistance to Endocrine Treatment; SRC-1 is an Independent Predictor of Breast Cancer Recurrence

This study investigates the role of the p160 coactivators AIB1 and SRC-1 independently, and their interactions with the estrogen receptor, in the development of resistance to endocrine treatments. The expression of the p160s and the estrogen receptor, and their interactions, was analyzed by immunohistochemistry and quantitative coassociation immunofluorescent microscopy, using cell lines, primary breast tumor cell cultures, and a tissue microarray with breast cancer samples from 560 patients. Coassociation of the p160s and estrogen receptor alpha was increased in the LY2 endocrine-resistant cell line following treatment with tamoxifen in comparison with endocrine-sensitive MCF-7 cells. In primary cultures, there was an increase in association of the coactivators with estrogen receptor alpha following estrogen treatment but dissociation was evident with tamoxifen. Immunohistochemical staining of the tissue microarray revealed that SRC-1 was a strong predictor of reduced disease-free survival (DFS), both in patients receiving adjuvant tamoxifen treatment and untreated patients (P < 0.0001 and P = 0.0111, respectively). SRC-1 was assigned a hazard ratio of 2.12 using a Cox proportional hazards model. Endocrine-treated patients who coexpressed AIB1 with human epidermal growth factor receptor 2 had a significantly shorter DFS compared with all other patients (P = 0.03). Quantitative coassociation analysis in the patient tissue microarray revealed significantly stronger colocalization of AIB1 and SRC-1 with estrogen receptor alpha in patients who have relapsed in comparison with those patients who did not recur (P = 0.026 and P = 0.00001, respectively). SRC-1 is a strong independent predictor of reduced DFS, whereas the interactions of the p160 proteins with estrogen receptor alpha can predict the response of patients to endocrine treatment.

Cancer Protective Action of Polysaccharide, Derived from Red Microalga Porphyridium Cruentum—A Biological Background

ABSTRACTThe cell wall sulfated polysaccharide of the red microalga Porphyridium cruentum (Rhodophyta) (PcrPSH) exhibited strong antitumor activity against Graffi myeloid tumor in hamsters both in vitro and in vivo. When tested in vivo, depending on the concentration and the way of application, this polysaccharide decreased transplantability in all experimental groups till 20 days of observation and mortality rate. The tumor growth was retarded and the mean survival time was prolonged with 10 - 16 days. Applied in in vitro experiments the PcrPSH increased both—spreading and phagocytic ability of peritoneal macrophages from healthy and Graffi tumor bearing hamsters in a dose—dependent manner. Primary Graffi tumor cell culture, cultivated in the presence of PcrPSH showed significant decrease of cell viability, determined by MTT test, while in cells derived from bone marrow it was increased at the same conditions of cultivation and concentration of polysaccharide. Primary Graffi tumor cell culture, treated with PcrPSH showed the appearance of a characteristic DNA ladder on agarose gel electrophoresis, which is a biochemical hallmark of apoptosis. The manifested anticancer activity of PcrPSH could be associated with its immunostimulating action as well as with direct cytotoxic properties. Based on these results, we could suggest that the tested algal PcrPSH is a promising candidate as an antitumor agent. Further studies will be done to clarify the mechanisms of a biological action of PcrPSH.

Heterogeneous in vitro effects of doxorubicin on gene expression in primary human liposarcoma cultures

BackgroundDoxorubicin is considered one of the most potent established chemotherapeutics in the treatment of liposarcoma; however, the response rates usually below 30%, are still disappointing. This study was performed to identify gene expression changes in liposarcoma after doxorubicin treatment.MethodsCells of 19 primary human liposarcoma were harvested intraoperatively and brought into cell culture. Cells were incubated with doxorubicin for 24 h, RNA was isolated and differential gene expression was analysed by the microarray technique.ResultsA variety of genes involved in apoptosis were up and down regulated in different samples revealing a heterogeneous expression pattern of the 19 primary tumor cell cultures in response to doxorubicin treatment. However, more than 50% of the samples showed up-regulation of pro-apoptotic genes such as TRAIL Receptor2, CDKN1A, GADD45A, FAS, CD40, PAWR, NFKBIA, IER3, PSEN1, RIPK2, and CD44. The anti-apoptotic genes TNFAIP3, PEA15, Bcl2A1, NGFB, and BIRC3 were also up-regulated. The pro-apoptotic CD14, TIA1, and ITGB2 were down-regulated in more than 50% of the tumor cultures after treatment with doxorubicin, as was the antiapoptotic YWHAH.ConclusionDespite a correlation of the number of differentially regulated genes to the tumor grading and to a lesser extent histological subtype, the expression patterns varied strongly; however, especially among high grade tumors the responses of selected apoptosis genes were similar. The predescribed low clinical response rates of low grade liposarcoma to doxorubicin correspond to our results with only little changes on gene expression level and also divergent findings concerning the up- and down-regulation of single genes in the different sarcoma samples.

Characterization of the cytotoxic activity of the indoloquinoline alkaloid cryptolepine in human tumour cell lines and primary cultures of tumour cells from patients

The plant derived indoloquinoline alkaloid cryptolepine was investigated for its cytotoxic properties in 12 human tumour cell lines and in primary cultures of tumour cells from patients. The fluorometric microculture cytotoxicity assay was used to assess cytotoxicity and DNA micro-array analysis to evaluate gene expression. Cryptolepine mean IC(50) in the cell line panel was 0.9 microM compared with 1.0 and 2.8 microM in haematological and solid tumour malignancies, respectively. Among patient solid tumour samples, those from breast cancer were the most sensitive and essentially as sensitive as haematological malignancies. Cryptolepine activity showed highest correlations to topoisomerase II and microtubule targeting drugs. In the cell lines cryptolepine activity was essentially unaffected by established mechanisms of drug resistance. A number of genes were identified as associated with cryptolepine activity. In conclusion, cryptolepine shows interesting in vitro cytotoxic properties and its further evaluation as an anti-cancer drug seems warranted.

Genomic and proteomic characterization of YDOV-157, a newly established human epithelial ovarian cancer cell line

The existence of several model systems with which to investigate a particular disease is advantageous for researchers. This is especially true for ovarian cancer, which, due to its complex and heterogeneous nature, inherently requires a large number of model systems. Here, we report a new ovarian serous adenocarcinoma cell line, designated YDOV-157, and characterized via post genomics and post proteomics. In this study, primary culture of tumor cells from ascites was performed and the cells were immortalized up to at least 60 passages in vitro. We studied the morphologies, cell proliferation, BRCA1/2 mutations, tumorigenesis capacity, and chemosensitivity of YDOV-157. Using a cDNA microarray, differentially expressed genes were identified and some of them were validated. Using proteomic analysis, we identified proteins that were differentially expressed in YDOV-157. The newly derived cell line, designated YDOV-157, grew as a monolayer and the doubling time was 102 h. When transplanted into nude mice, it initiated the formation of tumor masses with microscopic findings identical to those of the primary tumor. Chemosensitivity test showed that paclitaxel induced the highest chemosensitivity index. In microarray analysis, 2,520 probes were differently expressed, compared to human ovarian surface epithelial cells (HOSEs). In SYBR Green real-time PCR, the expression of E2F2 (P = 0.040) and CRABP2 genes (P = 0.030) was significantly higher in the ovarian cancer cell lines than in HOSEs. Furthermore, proteomic analysis showed that expression of 28 spots was significantly altered between YDOV-157 and HOSE. In conclusion, the newly derived YDOV-157 cell line may be an important research resource for studying cancer cell biology and should also be very useful for developing new strategies that inhibit cancer cell growth and progression.

Laser microdissection and primary cell cultures improve pharmacogenetic analysis in pancreatic adenocarcinoma

A key focus of research on pancreatic ductal adenocarcinoma (PDAC) is identifying new techniques to tailor gemcitabine and 5-fluorouracil treatments. Availability of tumor tissue is critical for the accurate assessment of gene expression, and laser microdissection (LMD) and primary cell cultures may be useful tools to separate tumor cells from the stromal reaction. The aim of this study was (1) to address the genetic profile relevant to drug activity and (2) to evaluate differences between microdissected and non-microdissected tumors, normal tissues, and primary cell cultures. Quantitative PCR of seven key genes was performed on mRNA from 113 microdissected and 28 non-microdissected tumors, a pool of normal tissues and four established primary cell lines. Protein expression was evaluated by western blot and immunocytochemistry and cytotoxicity by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. LMD allowed the analysis of 110 samples and revealed significant differences in mRNA levels between microdissected tumors and normal tissues, as well as between non-microdissected and microdissected tumors from the same patients. In contrast, primary cell lines showed similar expression profiles with respect to their respective microdissected tumors. In particular, expression levels of human equilibrative nucleoside transporter-1 and thymydilate synthase were significantly related to gemcitabine and 5-fluorouracil cytotoxicity. We conclude that LMD is a reliable technique for mRNA extraction, and allows detection of significant differences in the expression of specific target genes when compared to non-microdissected specimens and normal tissues. Moreover, expression levels in microdissected tumors are similar to those observed in primary tumor cell cultures, both at mRNA and protein level, and are related to drug chemosensitivity. The use of these ex vivo techniques for molecular analysis of tumors therefore appears to be of some value in implementing the clinical management of PDAC.

Altered localisation of the copper efflux transporters ATP7A and ATP7B associated with cisplatin resistance in human ovarian carcinoma cells

BackgroundCopper homeostasis proteins ATP7A and ATP7B are assumed to be involved in the intracellular transport of cisplatin. The aim of the present study was to assess the relevance of sub cellular localisation of these transporters for acquired cisplatin resistance in vitro. For this purpose, localisation of ATP7A and ATP7B in A2780 human ovarian carcinoma cells and their cisplatin-resistant variant, A2780cis, was investigated.MethodsSub cellular localisation of ATP7A and ATP7B in sensitive and resistant cells was investigated using confocal fluorescence microscopy after immunohistochemical staining. Co-localisation experiments with a cisplatin analogue modified with a carboxyfluorescein-diacetate residue were performed. Cytotoxicity of the fluorescent cisplatin analogue in A2780 and A2780cis cells was determined using an MTT-based assay. The significance of differences was analysed using Student's t test or Mann-Whitney test as appropriate, p values of < 0.05 were considered significant.ResultsIn the sensitive cells, both transporters are mainly localised in the trans-Golgi network, whereas they are sequestrated in more peripherally located vesicles in the resistant cells. Altered localisation of ATP7A and ATP7B in A2780cis cells is likely to be a consequence of major abnormalities in intracellular protein trafficking related to a reduced lysosomal compartment in this cell line. Changes in sub cellular localisation of ATP7A and ATP7B may facilitate sequestration of cisplatin in the vesicular structures of A2780cis cells, which may prevent drug binding to genomic DNA and thereby contribute to cisplatin resistance.ConclusionOur results indicate that alterations in sub cellular localisation of transport proteins may contribute to cisplatin resistance in vitro. Investigation of intracellular protein localisation in primary tumour cell cultures and tumour tissues may help to develop markers of clinically relevant cisplatin resistance. Detection of resistant tumours in patients may in turn enable individualization of the chemotherapy in the early stage of treatment.

Involvement of insulin‐like growth factor‐insulin receptor signal pathway in the transgenic mouse model of medulloblastoma

A transgenic mouse model expressing Simian virus 40 T-antigen (SV40Tag) under the control of a tetracycline system was generated. In this model, a cerebellar tumor was developed after doxycycline hydrochloride treatment. Real time-polymerase chain reaction and immunohistochemistry results indicated that the SV40Tag gene was expressed in the tumor. Pathological analysis showed that the tumor belonged to medulloblastoma. Further molecular characterization of the tumor demonstrated that the insulin-like growth factor (IGF) signaling pathway was activated. We also found that the SV40Tag could bind and translocate insulin receptor substrate 1 into the nucleus in primary cultured tumor cells. The interaction between the IGF pathway and SV40Tag may contribute to the process of malignant transformation in medulloblastoma. This transgenic animal model provides an important tool for studies on the signal pathways involved in the preneoplastic process in medulloblastoma and could help to identify therapeutic targets for brain tumors.

Defective DNA strand break repair causes chromosomal instability and accelerates liver carcinogenesis in mice

Chromosomal instability is a characteristic feature of hepatocellular carcinoma (HCC) but its origin and role in liver carcinogenesis are undefined. We tested whether a defect in the nonhomologous end-joining (NHEJ) DNA repair gene Ku70 was associated with chromosomal abnormalities and enhanced liver carcinogenesis. Male Ku70 NHEJ-deficient (Ku70-/-), heterozygote (Ku70 +/-), and wild-type (WT) mice were injected with diethylnitrosamine (DEN), a liver carcinogen, at age 15 days. Animals were killed at 3, 6, and 9 months for assessment of tumorigenesis and hepatocellular proliferation. For karyotype analysis, primary liver tumor cell cultures were prepared from HCCs arising in Ku70 mice of all genotypes. Compared to WT littermates, Ku70-/- mice injected with DEN displayed accelerated HCC development. Ku70-/- HCCs harbored clonal increases in numerical and structural aberrations of chromosomes 4, 5, 7, 8, 10, 14, and 19, many of which recapitulated the spectrum of equivalent chromosomal abnormalities observed in human HCC. Ku70-/- HCCs showed high proliferative activity with increased cyclin D1 and proliferating cell nuclear antigen expression, Aurora A kinase activity, enhanced ataxia telangiectasia mutated kinase and ubiquitination, and loss of p53 via proteasomal degradation, features which closely resemble those of human HCC. These findings demonstrate that defects in the NHEJ DNA repair pathway may participate in the disruption of cell cycle checkpoints leading to chromosomal instability and accelerated development of HCC.

The novel alkylating prodrug J1: diagnosis directed activity profile ex vivo and combination analyses in vitro

The dipeptide J1 acts as a prodrug of melphalan with a significant increased potency in vitro resulting from activation by cellular aminopeptidases. The current study was performed to evaluate the ex vivo profile of J1 using 176 primary tumor cell cultures from patients. In addition, the activity of J1 in combination with eight standard drugs, representing different mechanistic classes, was studied in nine different human tumor cell lines of different histopathological origin. Ex vivo evaluation of tumor type selectivity, was performed using the established fluorometric microculture cytotoxicity assay (FMCA). Combinations between J1 and eight standard chemotherapeutic drugs were analyzed using the median-effect method. The prodrug J1 expressed approximately 50- to 100-fold higher potency but similar activity profile as that of its metabolite, melphalan. The difference was greater in some diagnoses (e.g. breast cancer, NHL and AML), and exceptionally high in some breast cancer samples with aggressive phenotypes. Combination analysis of J1 and standard chemotherapeutics yielded several potentially additive and synergistic interactions, most striking for etoposide with significant synergism in all studied cell lines. In conclusion, the ex vivo profile suggests that further evaluation of J1 as the alkylating agent in for example aggressive breast cancer might be of particular interest, preferentially in combination with DNA-topoisomerase II inhibitors like etoposide.

Interferon-γ inhibits cellular proliferation and ACTH production in corticotroph tumor cells through a novel JAK-STAT1/NF-κB inhibitory signalling pathway

Cushing's disease is a severe clinical condition caused by hypersecretion of corticosteroids due to excessive ACTH secretion from a pituitary adenoma. In the last years there was only few progress elucidating the molecular mechanisms responsible for constitutive and autonomous ACTH secretion of pituitary corticotrophinomas. As a consequence, no effective drug therapy is currently available, particularly if surgical excision is not successful. IFN-γ is a cytokine that exerts potent antiproliferative and tumoricidal effects in a variety of cancers. Moreover, IFN-γ modulates normal pituitary hormone secretion, and has been shown to inhibit the expression of the precursor of ACTH, POMC, in murine ACTH secreting AtT-20 tumor cells. In order to identify molecular targets for the treatment of Cushing's disease we have studied the involvement of IFN-γ in the molecular events leading to the control of POMC transcriptional repression. We demonstrate that IFN-γ inhibits proliferation and POMC transcriptional activity of AtT-20 tumor cells via the activation of the JAK-STAT1 signalling cascade. Furthermore, the transcription factor NF-κB is associated with the transcriptional inhibition of POMC by IFN-γ. In addition, α and β IFN-γ receptor subunits are present at the protein level in human corticotroph tumor cells. Interestingly, IFN-γ inhibits ACTH production from human ACTH secreting tumor cells in primary cell culture without affecting basal ACTH biosynthesis in normal non-tumoral pituitary cells. In conclusion, our data for the first time show that POMC transcription can be negatively regulated by a JAK-STAT1 and NF-κB-dependent signalling pathway. The development of therapeutic agents that target this novel IFN-γ-JAK-STAT1/NF-κB pathway could therefore prove valuable for the effective treatment of Cushing's disease.

A Transgenic Mouse Model of Plasma Cell Malignancy Shows Phenotypic, Cytogenetic, and Gene Expression Heterogeneity Similar to Human Multiple Myeloma

Multiple myeloma is an incurable plasma cell malignancy for which existing animal models are limited. We have previously shown that the targeted expression of the transgenes c-Myc and Bcl-X(L) in murine plasma cells produces malignancy that displays features of human myeloma, such as localization of tumor cells to the bone marrow and lytic bone lesions. We have isolated and characterized in vitro cultures and adoptive transfers of tumors from Bcl-xl/Myc transgenic mice. Tumors have a plasmablastic morphology and variable expression of CD138, CD45, CD38, and CD19. Spectral karyotyping analysis of metaphase chromosomes from primary tumor cell cultures shows that the Bcl-xl/Myc tumors contain a variety of chromosomal abnormalities, including trisomies, translocations, and deletions. The most frequently aberrant chromosomes are 12 and 16. Three sites for recurring translocations were also identified on chromosomes 4D, 12F, and 16C. Gene expression profiling was used to identify differences in gene expression between tumor cells and normal plasma cells (NPC) and to cluster the tumors into two groups (tumor groups C and D), with distinct gene expression profiles. Four hundred and ninety-five genes were significantly different between both tumor groups and NPCs, whereas 124 genes were uniquely different from NPCs in tumor group C and 204 genes were uniquely different from NPCs in tumor group D. Similar to human myeloma, the cyclin D genes are differentially dysregulated in the mouse tumor groups. These data suggest the Bcl-xl/Myc tumors are similar to a subset of plasmablastic human myelomas and provide insight into the specific genes and pathways underlying the human disease.