Sen Pathak

Essential role of limiting telomeres in the pathogenesis of Werner syndrome

Mutational inactivation of the gene WRN causes Werner syndrome, an autosomal recessive disease characterized by premature aging, elevated genomic instability and increased cancer incidence. The capacity of enforced telomerase expression to rescue premature senescence of cultured cells from individuals with Werner syndrome and the lack of a disease phenotype in Wrn-deficient mice with long telomeres implicate telomere attrition in the pathogenesis of Werner syndrome. Here, we show that the varied and complex cellular phenotypes of Werner syndrome are precipitated by exhaustion of telomere reserves in mice. In late-generation mice null with respect to both Wrn and Terc (encoding the telomerase RNA component), telomere dysfunction elicits a classical Werner-like premature aging syndrome typified by premature death, hair graying, alopecia, osteoporosis, type II diabetes and cataracts. This mouse model also showed accelerated replicative senescence and accumulation of DNA-damage foci in cultured cells, as well as increased chromosomal instability and cancer, particularly nonepithelial malignancies typical of Werner syndrome. These genetic data indicate that the delayed manifestation of the complex pleiotropic of Wrn deficiency relates to telomere shortening.

LNCaP progression model of human prostate cancer: Androgen‐independence and osseous metastasis

Clinically, the lethal phenotypes of human prostate cancer are characterized by their progression to androgen‐independence and their propensity to form osseous metastases. We reported previously on the establishment of androgen‐independent (AI) human prostate cancer cell lines derived from androgen‐dependent (AD) LNCaP cells, with androgen independence defined as the capability of prostate cancer cells to grow in castrated hosts. One of the sublines, C4‐2, was found to be AI, highly tumorigenic, and metastatic, having a proclivity for metastasis to the bone.

Telomere dysfunction suppresses spontaneous tumorigenesis in vivo by initiating p53‐dependent cellular senescence

Dysfunctional telomeres induce p53‐dependent cellular senescence and apoptosis, but it is not known which function is more important for tumour suppression in vivo. We used the p53R172P knock‐in mouse, which is unable to induce apoptosis but retains intact cell‐cycle arrest and cellular senescence pathways, to show that spontaneous tumorigenesis is potently repressed in Terc−/−p53R172P mice. Tumour suppression is accompanied by global induction of p53, p21 and the senescence marker senescence‐associated‐β‐galactosidase. By contrast, cellular senescence was unable to suppress chemically induced skin carcinomas. These results indicate that suppression of spontaneous tumorigenesis by dysfunctional telomeres requires the activation of the p53‐dependent cellular senescence pathway.

Isolation and Characterization of Metastatic Variants from Human Transitional Cell Carcinoma Passaged by Orthotopic Implantation in Athymic Nude Mice

PURPOSE These studies were designed to develop an orthotopic model for human bladder cancer and to isolate variant metastatic cell lines. MATERIALS AND METHODS The human bladder cancer cell line 253J was implanted into the muscular wall of the bladder of athymic nude mice. By in vivo recycling, we selected for 2 variant cell lines: 253J B-V, a bladder line isolated after 5 serial passages in the bladder, and 253J lung-IV, established from a lung tumor nodule that was recycled through the bladder. RESULTS These 2 cell lines showed enhanced tumorigenicity, as measured by a decreased latent period, and rapid growth as compared with the parental cell line. Moreover, orthotopic implantation of these cell lines resulted in metastasis to the lungs. These in vivo-selected, metastatic cell lines exhibited unique karyotypic alterations, increased anchorage-independent growth, overexpression of basic fibroblast growth factor, altered expression of adhesion molecules and the ability to migrate through Matrigel. CONCLUSIONS This reproducible model of human bladder cancer offers the opportunity to study cellular properties associated with tumor progression and metastasis and is suitable for the evaluation of new therapeutic strategies for invasive bladder cancer.

Tumor-Specific Low Molecular Weight Forms of Cyclin E Induce Genomic Instability and Resistance to p21, p27, and Antiestrogens in Breast Cancer

The deregulated expression of cyclin E as measured by the overexpression of its low molecular weight (LMW) isoforms is a powerful predictor of poor outcome in patients with breast cancer. The mechanism by which these LMW forms give tumor cells a growth advantage is not known and is the subject of this article. In this article, we provide the pathobiological mechanisms of how these LMW forms are involved in disease progression. Specifically, we show that overexpression of the LMW forms of cyclin E but not the full-length form in MCF-7 results in (a) their hyperactivity because of increased affinity for cdk2 and resistance to inhibition by the cyclin-dependent kinase inhibitors p21 and p27, (b) resistance to the growth inhibiting effects of antiestrogens, and (c) chromosomal instability. Lastly, tumors from breast cancer patients overexpressing the LMW forms of cyclin E are polyploid in nature and are resistant to endocrine therapy. Collectively, the biochemical and functional differences between the full-length and the LMW isoforms of cyclin E provide a molecular mechanism for the poor clinical outcome observed in breast cancer patients harboring tumors expressing high levels of the LMW forms of cyclin E. These properties of the LMW forms cyclin E suggest that they are not just surrogate markers of poor outcome but bona fide mediators of aggressive disease and potential therapeutic targets for patients whose tumors overexpress these forms.

Coevolution of Prostate Cancer and Bone Stroma in Three-Dimensional Coculture: Implications for Cancer Growth and Metastasis

Human bone stromal cells, after three-dimensional coculture with human prostate cancer (PCa) cells in vitro, underwent permanent cytogenetic and gene expression changes with reactive oxygen species serving as mediators. The evolved stromal cells are highly inductive of human PCa growth in mice, and expressed increased levels of extracellular matrix (versican and tenascin) and chemokine (BDFN, CCL5, CXCL5, and CXCL16) genes. These genes were validated in clinical tissue and/or serum specimens and could be the predictors for invasive and bone metastatic PCa. These results, combined with our previous observations, support the concept of permanent genetic and behavioral changes of PCa epithelial cells after being either cocultured with prostate or bone stromal cells as three-dimensional prostate organoids or grown as tumor xenografts in mice. These observations collectively suggest coevolution of cancer and stromal cells occurred under three-dimensional growth condition, which ultimately accelerates cancer growth and metastasis.

POT1b protects telomeres from end-to-end chromosomal fusions and aberrant homologous recombination

POT1 (protection of telomere 1) is a highly conserved single‐stranded telomeric binding protein that is essential for telomere end protection. Here, we report the cloning and characterization of a second member of the mouse POT family. POT1b binds telomeric DNA via conserved DNA binding oligonucleotide/oligosaccharide (OB) folds. Compared to POT1a, POT1b OB‐folds possess less sequence specificity for telomeres. In contrast to POT1a, truncated POT1b possessing only the OB‐folds can efficiently localize to telomeres in vivo. Overexpression of a mutant Pot1b allele that cannot bind telomeric DNA initiated a DNA damage response at telomeres that led to p53‐dependent senescence. Furthermore, a reduction of the 3′ G‐rich overhang, increased chromosomal fusions and elevated homologous recombination (HR) were observed at telomeres. shRNA mediated depletion of endogenous Pot1b in Pot1a deficient cells resulted in increased chromosomal aberrations. Our results indicate that POT1b plays important protective functions at telomeres and that proper maintenance of chromosomal stability requires both POT proteins.

Orthotopic implantation is essential for the selection, growth and metastasis of human real cell cancer in nude mice

Human neoplasms are heterogeneous for a variety of biological properties that include invasion and metastasis. The presence of a small subpopulation of cells with a highly metastatic phenotype has important clinical implications for diagnosis and therapy of cancer. For this reason, it is important to develop an animal model for the selection and isolation of metastatic variants from human neoplasms and for testing the metastatic potential of human tumor cells.We have implanted human renal cell carcinoma (HRCC) cells (obtained from a surgical specimen) into different organs of nude mice and then recovered the tumors and established each in culture. The 5 established lines differed in their biological-metastatic properties and had a unique karyotype, indicating that growth at different organs selects for different subpopulations of HRCC. Moreover, the HRCC did not metastasize unless they were implanted orthotopically. These findings indicate that the appropriate nude mouse model for studying the biology and therapy of HRCC must be based on the orthotopic implantation of tumor cells.

Adenosine acts as an inhibitor of lymphoma cell growth: a major role for the A3 adenosine receptor.

In this study, we demonstrated several mechanisms exploring the inhibitory effect of low-dose adenosine on lymphoma cell growth. Adenosine, a purine nucleoside present in plasma and other extracellular fluids, acts as a regulatory molecule, by binding to G-protein associated cell-surface receptors, A1, A2 and A3. Recently we showed that low-dose adenosine released by muscle cells, inhibits tumour cell growth and thus attributes to the rarity of muscle metastases. In the present work, a cytostatic effect of adenosine on the proliferation of the Nb2-11C rat lymphoma cell line was demonstrated. This effect was mediated through the induction of cell cycle arrest in the G0/G1 phase and by decreasing the telomeric signal in these cells. Adenosine was found to exert its antiproliferative effect mainly through binding to its A3 receptor. The cytostatic anticancer activity, mediated through the A3 adenosine receptor, turns it into a potential target for the development of anticancer therapies.

PERMANENT PHENOTYPIC AND GENOTYPIC CHANGES OF PROSTATE CANCER CELLS CULTURED IN A THREE-DIMENSIONAL ROTATING-WALL VESSEL

SummaryA three-dimensional (3D) integrated rotating-wall vessel cell-culture system was used to evaluate the interaction between a human prostate cancer cell line, LNCaP, and microcarrier beads alone, or microcarrier beads previously seeded with either prostate or bone stromal cells. Upon coculture of LNCaP cells with microcarrier beads either in the presence or in the absence of prostate or bone stromal cells, 3D prostate organoids were formed with the expected hormonal responsiveness to androgen, increased cell growth, and prostate-specific antigen production. In this communication, we define permanent phenotypic and genotypic changes of LNCaP cells upon coculture with microcarrier beads alone, or with microcarrier beads previously seeded with either prostate or bone stromal cells. Most notably, we observed selective genetic changes, i.e., chromosomal losses or gains, as evaluated by both conventional cytogenetic and comparative genomic hybridization, in LNCaP sublines derived from the prostate organoids. Moreover, the derivative LNCaP cells appear to have altered growth profiles, and exhibit permanent and stable changes in response to androgen, estrogen, and growth factors. The derivative LNCaP sublines showed increased anchorage-independent growth rate, and enhanced tumorigenicity and metastatic potential when inoculated orthotopically in castrated athymic mice. Our results support the hypothesis that resembles “adaptive mutation” such as has been described in bacteria subjected to nutritional starvation. The occurrence of such permanent changes may be highly contact dependent, and appears to be driven by specific microenvironmental factors surrounding the tumor cell epithelium grown as 3D prostate organoids.