Search PubMed⌕ Search

Biomedical subjects

Joseph R Testa

Publications and source records attributed to Joseph R Testa.

44 records · Page 3Linked to original sources
PubMed2002

SV40 infection induces telomerase activity in human mesothelial cells.

Mesotheliomas are malignant tumors of the pleural and peritoneal membranes which are often associated with asbestos exposure and with Simian virus 40 (SV40) infection. Telomerase activity is repressed in somatic cells and tissues but is activated in immortal and malignant cells. We evaluated telomerase activity in seven primary malignant mesothelioma biopsies and matched lung specimens and 20 mesothelioma cell lines and eight corresponding primary tumor cultures. All the tumor biopsies, and nearly all primary cell mesothelioma cultures and cell lines were telomerase positive. The findings in cell lines paralleled those observed in primary cultures in cases where paired samples were available. Next, we found that SV40, a DNA tumor virus present in approximately 50% of mesothelioma biopsies in the USA, induced telomerase activity in primary human mesothelial cells, but not in primary fibroblasts. Telomerase activity became detectable as early as 72 h following wild-type (strain 776) SV40 infection, and a clear DNA ladder was detectable 1 week after infection. The amount of telomerase activity increased during passage in cell culture and appeared to parallel increases in the cellular amounts of the SV40 large T-antigen. Thus, SV40 infection leads to telomerase activity before the infected mesothelial cells become transformed and immortalized. SV40 infection of human fibroblasts did not cause detectable telomerase activity. We also determined that the SV40 small t-antigen (tag) plays an important role in inducing telomerase activity because this activity was undetectable or minimal in mesothelial cells infected and/or transformed by SV40 tag mutants. Asbestos alone did not induce telomerase activity, and asbestos did not influence telomerase activity in mesothelial cells infected with SV40. Induction of telomerase activity by SV40 may be related to the very high rate of mesothelial cell immortalization that is characteristically associated with SV40 infection of mesothelial cells.

Blotting, Southern↗
PubMed2002

Frequent activation of AKT2 kinase in human pancreatic carcinomas.

Activation of AKT/protein kinase B promotes a variety of biological activities important in tumorigenesis, such as cell survival and cell cycle progression. We previously demonstrated amplification and overexpression of the AKT2 gene in a subset of human pancreatic carcinomas. In this investigation, we assessed AKT2 catalytic activity in 50 frozen pancreatic tissues (37 carcinomas, four benign tumors and nine normal pancreata) by in vitro kinase assay. Twelve of 37 (32%) pancreatic carcinomas showed markedly elevated levels of AKT2 activity compared to normal pancreata and begin pancreatic tumors. To delineate mechanisms contributing to AKT2 activation in malignant pancreatic tumors, we examined the status of upstream components of the phosphatilydlinositol 3-kinase (PI3K)/AKT pathway. Western blot analysis revealed loss of PTEN protein expression in two of the 12 pancreatic carcinomas with activated AKT2. In vitro PI3K assays demonstrated high levels of PI3K activity in seven carcinoma specimens that showed AKT2 activation. Immunohistochemical staining confirmed high levels of phosphorylated (active) AKT in malignant pancreatic tumors compared to normal pancreata. Overall, these data suggest that upstream perturbations of the PI3K/AKT pathway contribute to frequent activation of AKT2 in pancreatic cancer, which may contribute to the pathogenesis of this highly aggressive form of human malignancy.

Adenocarcinoma↗
PubMed2002

The pathogenesis of mesothelioma.

About 80% of malignant mesotheliomas (MM) in the Western World develop in individuals with higher than background exposure to asbestos. Only a fraction of those exposed to asbestos develop mesothelioma, indicating that additional factors play a role. Simian virus 40 (SV40), a DNA tumor virus that preferentially causes mesothelioma in hamsters, has been detected in several human mesotheliomas. The expression of the SV40 large tumor antigen in mesothelioma cells, and not in nearby stromal cells, and the capacity of antisense T-antigen treatment to arrest mesothelioma cell growth in vitro suggest that SV40 contributes to tumor development. The capacity of T-antigen to bind and inhibit cellular p53 and retinoblastoma (Rb)-family proteins in mesothelioma, together with the very high susceptibility of human mesothelial cells to SV40-mediated transformation in vitro, supports a causative role of SV40 in the pathogenesis of mesothelioma. Asbestos appears to increase SV40-mediated transformation of human mesothelial cells in vitro, suggesting that asbestos and SV40 may be cocarcinogens. p53 mutations are rarely found in mesothelioma; p16, p14ARF, and NF2 mutations/losses are frequent. Recent studies revealed the existence of a genetic factor that predisposes affected individuals to mesothelioma in the villages of Karain and Tuzkoy, in Anatolia, Turkey. Erionite, a type of zeolite, may be a cofactor in these same villages, where 50% of deaths are caused by mesothelioma. Mesothelioma appears to have a complex etiology in which environmental carcinogens (asbestos and erionite), ionizing radiation, viruses, and genetic factors act alone or in concert to cause malignancy.

Animals↗
PubMed2002

Akt1 and Akt2 differently regulate muscle creatine kinase and myogenin gene transcription in insulin-induced differentiation of C2C12 myoblasts.

Insulin and IGFs are potent inducers of skeletal muscle differentiation. Although PI3K is known to be involved in skeletal muscle differentiation, its downstream targets in this process are not clearly defined. We investigated the roles of Akt and mammalian target of rapamycin (mTOR) in skeletal muscle differentiation. LY294002, a pharmacological inhibitor of PI3K, and the immunosuppressant rapamycin inhibited insulin-induced differentiation of C2C12 myoblasts. LY294002 and rapamycin suppressed myosin heavy chain expression and myotube formation. Transient reporter assays showed that both inhibitors repress muscle creatine kinase (MCK) and myogenin gene transcription. Heterologous expression of Akt1/PKB(alpha) potently suppressed MCK gene transcription without affecting myogenin gene transcription, whereas heterologous expression of Akt2 increased myogenin and MCK gene transcription. Finally, overexpression of myogenin rescued the inhibitory effect of rapamycin on MCK gene transcription, whereas it failed to rescue the inhibitory effect of LY294002 and Akt1. These results suggest that insulin regulates myogenic differentiation chiefly at the level of myogenin gene transcription via PI3K and mTOR. PI3K activity, but not mTOR, may regulate transcriptional activity of myogenin. Our data also suggest that Akt1 and Akt2 play distinct roles in myogenic differentiation.

Animals↗
PubMed2002

Detection of SV40 DNA sequences in malignant mesothelioma specimens from the United States, but not from Turkey.

The incidence of malignant mesothelioma (MM) shows a strong epidemiological association with exposure to asbestos fibers. Recently, simian virus 40 (SV40) DNA sequences have been reported in MM tumor specimens from the United States and several European countries, and the SV40 tumor virus has been implicated as a potential co-factor in the etiology of this disease. However, several large studies from the US, Finland, and Turkey did not detect SV40 sequences in MM samples. To address this discrepancy, MM specimens from Turkey and the US were analyzed in the same laboratory under identical conditions to detect the presence of SV40 DNA. We detected SV40 sequences in 4 of 11 specimens from the United States, but in none of the 9 Turkish samples examined. These findings suggest that geographical differences exist with regard to the involvement of SV40 in human tumors.

Blotting, Southern↗
PubMed2001

p21-activated kinase links Rac/Cdc42 signaling to merlin.

The neurofibromatosis type 2 tumor suppressor gene, NF2, is mutated in the germ line of NF2 patients and predisposes affected individuals to intracranial and spinal tumors. Moreover, somatic mutations of NF2 can occur in the sporadic counterparts of these neurological tumor types as well as in certain neoplasms of non-neuroectodermal origin, such as malignant mesothelioma and melanoma. NF2 encodes a 595-amino acid protein, merlin, which exhibits significant homology to the ezrin-radixin-moesin family of proteins. However, the mechanism by which merlin exerts its tumor suppressor activity is not well understood. In this investigation, we show that merlin is phosphorylated in response to expression of activated Rac and activated Cdc42 in mammalian cells. Furthermore, we demonstrate that merlin phosphorylation is mediated by p21-activated kinase (Pak), a common downstream target of both Rac and Cdc42. Both in vivo and in vitro kinase assays demonstrated that Pak can directly phosphorylate merlin at serine 518, a site that affects merlin activity and localization. These biochemical investigations provide insights into the regulation of merlin function and establish a framework for elucidating tumorigenic mechanisms involved in neoplasms associated with merlin inactivation.

3T3 Cells↗
PubMedDate not supplied

A clue to the therapy of neurofibromatosis type 2: NF2/merlin is a PAK1 inhibitor.

BACKGROUND: Neurofibromatosis type 2 is a group of tumors caused by loss-of-function mutations of a tumor suppressor gene encoding NF2/merlin. Development of chemotherapeutics for this disease, which often threatens the life of young children, has been hampered by a limited information on the signaling function of NF2. NF2 can inhibit Ras-induced malignant transformation. However, the primary (signaling) target of NF2 in the oncogenic pathway has not been previously identified. RESULTS: Here, using a series of NF2 constructs, we show that NF2 inhibits directly the Rac/CDC42-dependent Ser/Thr kinase PAK1, which is essential for both Ras transformation and neurofibromatosis type 1 (NF1), through two separate domains. A mutant of NF2, that lacks the PAK1-inhibiting domain of 78 amino acids (NF78C, residues 447-524), fails to suppress Ras transformation. Furthermore, PAK1-specific inhibitors CEP-1347 and WR-PAK18 selectively inhibit the growth of NF2-deficient cancer cells, but not NF2-positive cells. CONCLUSIONS: These results suggest that PAK1 is essential for the malignant growth of NF2-deficient cells, and that PAK1-blocking drugs could be potentially useful forthe treatment of neurofibromatosis types 2, in addition to Ras-induced cancers and neurofibromatosis type 1.

Animals↗