Risk and preventive factors for cot death in The Netherlands, a low-incidence country

BACKGROUND: The p53 tumor suppressor gene is mutated in up to 70% of pancreatic adenocarcinomas. We determined the effect of reintroduction of the wild-type p53 gene on proliferation and apoptosis in human pancreatic cancer cells using an adenoviral vector containing the wild-type p53 tumor suppressor gene. METHODS: Transduction efficiencies of six p53-mutant pancreatic cancer cell lines (AsPC-1, BxPC-3, Capan-1, CFPAC-1, MIA PaCa-2, and PANC-1) were determined using the reporter gene construct Ad5/CMV/beta-gal. Cell proliferation was monitored using a 3H-thymidine incorporation assay, Western blot analysis for p53 expression was performed, and DNA laddering and fluorescence-activated cell sorter analysis were used to assess apoptosis. p53 gene therapy was tested in vivo in a subcutaneous tumor model. RESULTS: The cell lines varied in transduction efficiency. The MIA PaCa-2 cells had the highest transduction efficiency, with 65% of pancreatic tumor cells staining positive for beta-galactosidase (beta-gal) at a multiplicity of infection (MOI) of 50. At the same MOI, only 15% of the CFPAC-1 cells expressed the beta-gal gene. Adenovirus-mediated p53 gene transfer suppressed growth of all human pancreatic cancer cell lines in a dose-dependent manner. Western blot analysis confirmed the presence of the p53 protein product at 48 hours after infection. DNA ladders demonstrated increased chromatin degradation, and fluorescence-activated cell sorter analysis demonstrated a four-fold increase in apoptotic cells at 48 and 72 hours following infection with Ad5/CMV/p53 in the MIA PaCa-2 and PANC-1 cells. Suppression of tumor growth mediated by induction of apoptosis was observed in vivo in an established nude mouse subcutaneous tumor model following intratumoral injections of Ad5/CMV/p53. CONCLUSIONS: Introduction of the wild-type p53 gene using an adenoviral vector in pancreatic cancer with p53 mutations induces apoptosis and inhibits cell growth. These data provide preliminary support for adenoviral mediated p53 tumor suppressor gene therapy of human pancreatic cancer.     

GOK: a gene at 11p15 involved in rhabdomyosarcoma and rhabdoid tumor development

The expression of GOK, a gene recently identified at 11p15.5, was studied in breast cancer, rhabdomyosarcoma, and rhabdoid tumor cell lines. In these neoplasms, deletions at 11p15 and suppression of tumorigenicity induced by a normal human chromosome 11 were previously demonstrated. Whereas breast cancer cell lines express readily detectable levels of GOK mRNA, expression is absent in rhabdomyosarcoma and rhabdoid tumor cell lines. This is in contrast with the high expression of GOK in skeletal muscle, the normal tissue of origin of rhabdomyosarcomas, suggesting that down-regulation of GOK expression could be involved in tumor development. In agreement with this hypothesis, transfection of GOK cDNA into G401 derived from a rhabdoid tumor and RD cells derived from a rhabdomyosarcoma that do not express detectable levels of GOK mRNA, induced cell death. Because GOK expression is not compatible with growth of these tumor cells, these results support the hypothesis that loss of GOK expression plays a role in tumor establishment or progression and suggest that GOK may act as a recessive tumor suppressor gene in rhabdomyosarcomas and rhabdoid tumors. On the contrary, transfection of GOK cDNA into the breast cancer cell line HBL100 produced no detectable effects, indicating that the growth-suppressive effect of GOK in RD and G401 cells was specific. Because rhabdomyosarcomas have been observed in cases of Beckwith-Wiedemann syndrome, a genetic disorder linked to 11p15, a role of GOK in this disease cannot be excluded.     

IL-15 enhances the response of human gamma delta T cells to nonpeptide [correction of nonpetide] microbial antigens

Human gamma delta T cells have the ability to rapidly expand and produce IFN-gamma in response to nonpeptide Ags of microbial pathogens, in particular a class of compounds known as the prenyl phosphates. We investigated the ability of IL-15, a T cell growth factor, to modulate prenyl phosphate-induced gamma delta T cell proliferation and cytokine production. IL-15 significantly enhanced the expansion of gamma delta T cells in the peripheral blood after stimulation in vitro with isopentenyl pyrophosphate. Moreover, using gamma delta T cell clones, we determined that IL-15-induced T cell proliferation was dependent on the IL-2R beta chain but not the IL-2R alpha chain. We therefore studied the IL-15R alpha chain expression in human gamma delta T cells in the presence or absence of nonpeptide Ags. We found IL-15R alpha mRNA expression in IL-15-stimulated and Ag-stimulated human gamma delta T cells but not in resting gamma delta T cells. Although IL-15 itself had little effect on the production of IFN-gamma, IL-15 plus IL-12 acted synergistically to augment IFN-gamma production by gamma delta T cells. Moreover, we showed that this increase in IFN-gamma could be explained by the dual activation of STAT1 and STAT4 by IL-15 and IL-12, respectively. Taken together, these results suggest that IL-15 may contribute to activation of human gamma delta T cells in the immune response to microbial pathogens.     

Defective interleukin six expression and responsiveness in human mammary cells transformed by an adeno 5/SV40 hybrid virus

Mammary epithelial cells (MECs) were isolated and cultured from mammary glands of healthy women undergoing reduction mammoplasty. Normal MECs were infected with the transforming hybrid virus adeno-5/SV40. Two transformed epithelial cell lines, M1 and M2, were obtained, characterised phenotypically and studied for the production of and the response to cytokines and growth regulators. In both cell lines, expression of the SV40 large T antigen was associated with loss of interleukin 6 (IL-6) production and responsiveness as well as with down-regulation of IL-8 and transforming growth factor (TGF)-alpha production. Both M1 and M2 cell lines were capable of forming colonies in semisolid media, but upon injection into severe combined immunodeficient (SCID) mice only M2 cells were tumorigenic. DNA synthesis in M1 cells was partially inhibited by serum or TNF-alpha and weakly stimulated by hydrocortisone (HC) and IL-8. In contrast, M2 cells were totally unresponsive to a variety of growth regulators. Both lines overexpressed the p53 protein at levels about 20-fold higher than those observed in primary MEC cultures, but no mutations of the p53 gene could be detected. The date confirm the view that the expression in human mammary cells of different oncogenes - including the SV40 T antigen - is frequently associated with alterations of cytokine production and responsiveness.     

Relationship between p21 expression and mutation of the p53 tumor suppressor gene in normal and malignant ovarian epithelial cells

In many cell types, p53-mediated growth inhibition is dependent on induction of p21, which is an inhibitor of cyclin-dependent kinases that are required for cell cycle progression. Failure of mutant p53 proteins to transactivate p21 may lead to uncontrolled proliferation. Because many ovarian cancers have mutations in the p53 gene, we examined p21 levels in normal and malignant ovarian epithelial cells to determine whether p21 expression is dependent on wild-type p53. Normal ovarian epithelial cells and two ovarian cancer cell lines with wild-type p53 expressed readily detectable levels of p21, whereas in p53 null and mutant cell lines, expression of p21 was diminished strikingly. A correlation between the status of the p53 gene and p21 expression also was noted in 23 primary epithelial ovarian cancers. Normal levels of p21 RNA were seen in 4/7 (57%) cancers with wild-type p53, whereas 14/16 (88%) cancers with mutant p53 had reduced p21 expression (P < 0.05). In addition, we found that lambda-irradiation of normal and malignant ovarian epithelial cells with wild-type, but not mutant, p53 resulted in induction of p21. These data are suggestive that induction of p21 is a feature of p53-mediated growth inhibition in normal ovarian epithelial cells. Conversely, mutation of the p53 gene in ovarian cancers usually is associated with decreased p21 expression. The lack of an absolute correlation between p21 expression and the status of the p53 gene in ovarian cancers is consistent with other studies that have suggested that p21 may also be regulated by p53-independent pathways.     

Prenatal diagnosis and fetopathological findings in a fetus with ring chromosome 18

Aging is associated with a decline in T cell proliferative responses and aberrations in cytokine production. In the present study, we examined if aging might alter the expression of the tumor-suppressor protein p53 and the retinoblastoma susceptibility gene product (Rb) as well as the levels of Bcl-2 in resting and activated human T cells. No significant differences were observed in the basal levels of p53 protein among resting T cells from young and elderly humans. After stimulation with anti-CD3 monoclonal antibody (mAb) OKT3 and phorbol myristate acetate (PMA), T cells from young humans exhibited severalfold increases in p53 protein expression compared with resting T cells. By contrast, T cells from a substantial portion of elderly humans failed to demonstrate significant increases in p53 in response to anti-CD3 plus PMA. No age-related alterations in the levels of Rb or Bcl-2 proteins were observed in resting or anti-CD3/PMA-stimulated T cells. To delineate whether the age-related reductions in p53 expression might be linked to decreased interleukin-2 (IL-2) production, we compared the expression of p53 and IL-2 in anti-CD3/PMA-stimulated T cells from elderly people. The results showed that impaired induction of p53 expression in activated T cells from certain elderly people could be observed without considerable impairments in IL-2 production. These observations suggest that age-related reductions in T cell expression of p53 may contribute to the decline of T cell competence independent of the impairments in IL-2 production.     

Down-regulation of murine fibrosarcoma transforming growth factor-beta 1 expression by interleukin 7

BACKGROUND: Cytokine genes encode proteins that modulate immune system responses. Modification of tumor cells by the introduction of cytokine genes has been used as a strategy to augment host immunity. Interleukin 7 (IL-7) gene transfer enhances the immune response to tumor cells and can result in tumor regression. Transforming growth factor-beta 1 (TGF-beta 1) is a potent immunosuppressive cytokine produced by many tumors. We have previously reported that recombinant IL-7 decreases the expression of TGF-beta 1 by murine macrophages. PURPOSE: This study investigates the inhibition of tumor-derived TGF-beta 1 production as a possible mechanism for the enhanced antitumor immunity that accompanies IL-7 gene transfer. METHODS: A fibrosarcoma cell line (FSA-JmIL-7) genetically modified to produce IL-7 was used to evaluate the effects of IL-7 on tumor production of TGF-beta 1. The control cell line (FSA-Jneo) originated from the same parental fibrosarcoma cell line (FSA) and was produced by transduction with the same retroviral vector without the IL-7 gene. FSA-Jneo and FSA-JmIL-7 tumor cells were evaluated for the expression of TGF-beta 1 messenger RNA (mRNA). To determine if the observed change in TGF-beta 1 mRNA was associated with an alteration in protein secretion, we compared supernatants from tumor cell cultures for TGF-beta 1 production. Specific anti-TGF-beta 1 monoclonal antibody (MAb) was used to confirm the role of TGF-beta 1 in these assays. RESULTS: Compared with FSA parental and FSA-Jneo cells, FSA-JmIL-7 cells expressed TGF-beta 1 mRNA at a lower level. Compared with supernatants from FSA-Jneo cells, FSA-JmIL-7 supernatants contained consistently lower levels of TGF-beta 1 activity (P < .05). In addition, FSA-Jneo supernatants suppressed lymphocyte proliferation to a significantly greater degree than supernatants from FSA-JmIL-7 cells (P < .05). Studies with anti-TGF-beta 1 MAb added to the supernatants confirmed the role of TGF-beta 1 in inhibition of lymphocyte proliferation. CONCLUSION: These findings suggest that IL-7 gene transfer inhibits the production of TGF-beta 1 by tumor cells and thus may enhance the efficacy of the host's antitumor immune response. IMPLICATION: The regulation of endogenous tumor-derived cytokines in response to cytokine gene transfer may contribute to altered immune responses in the tumor microenvironment and thus may be an important additional parameter to assess in gene therapy.     

HLA class I-specific inhibitory receptors in human T lymphocytes: interleukin 15-induced expression of CD94/NKG2A in superantigen- or alloantigen-activated CD8+ T cells

A fraction of human T lymphocytes, predominantly CD8+, express receptors for HLA class I molecules typical of natural killer cells (natural killer receptors or NKRs) that inhibit T cell receptor-mediated functions. Herein, we analyzed possible mechanism(s) leading to the expression of NKRs by T cells responding to superantigens or allogeneic cells in vitro. We show that, in the presence of interleukin 15 (IL-15), T cells (depleted of NKR+ cells) responding to toxic shock syndrome toxin 1 de novo express CD94, a molecule that is part of a heterodimeric NKR with a broad specificity for different HLA class I alleles. Maximal CD94 expression occurred when IL-15 was added shortly after the cells were placed into culture, and CD94 expression started 4-6 days after addition of IL-15. Although both CD4+ and CD8+ cells expressed CD94, the simultaneous expression of NKG2A (i.e., the other component of the CD94/NKG2A inhibitory NKR) was confined to CD8+ cells. Similar data were obtained in T cell populations activated in mixed lymphocyte cultures in the presence of IL-15. The expression of CD94/NKG2A led to an impairment of allo-specific cytolytic activity by mixed lymphocyte culture-derived T cell populations or clones. Remarkably, cytolysis could be restored by the addition of anti-CD94 mAb, i.e., by masking the inhibitory NKRs.     

Altered expression of the tumor suppressor/oncoprotein p53 in SV40 Tag-transformed human placental trophoblast and malignant trophoblast cell lines

The expression of the tumor suppressor/oncoprotein p53 has been investigated in normal human placental villous trophoblast, in vitro propagated invasive extravillous trophoblast, SV40 tumor antigen (Tag)-immortalized extravillous trophoblast, human cytomegalovirus (hCMV)-infected syncytiotrophoblast and malignant trophoblast (choriocarcinoma) cell lines (JAR, JEG-3 and BeWo) using quantitative enzyme-linked immunosorbent assay (ELISA) and Western immunoblot methods using monoclonal antibodies specific for wild-type and mutant p53. The normal villous and extravillous trophoblast cells expressed low levels of the wild-type p53 protein, whereas normal terminally differentiated multinucleated syncytiotrophoblast cells, as well as hCMV-infected syncytiotrophoblast, showed a higher expression of the wild-type p53 protein. SV40 Tag-immortalized invasive trophoblast cells also showed a high expression of the wild-type p53 protein which remained complexed with the Tag protein. All the choriocarcinoma cell lines over expressed the mutant form of the p53 protein. The increased expression of p53 protein in the SV40 Tag-immortalized invasive trophoblast and choriocarcinoma cells paralleled with increased expression of the mouse double minute 2 (mdm2) oncogenic protein. Transforming growth factor (TGF)-beta inhibited proliferation of normal extravillous trophoblast cells. The antiproliferative effects of TGF-beta were reduced in SV40 Tag-immortalized cells and non-detectable in choriocarcinoma cell lines JAR, BeWo and JEG-3. The inactivation of p53 owing to complexing with Tag in the immortalized premalignant trophoblast and p53 mutation in the malignant trophoblast may be responsible for their aberrant proliferation and refractoriness to antiproliferative effects of TGF-beta observed in these cells as compared to the normal trophoblast. These results may suggest the role of p53 protein in trophoblast differentiation, transformation and tumorigenesis.     

Cytotoxic effects of adenovirus-mediated wild-type p53 protein expression in normal and tumor mammary epithelial cells

To evaluate the effects of the wild-type p53 expression in normal and tumor cells, we have constructed a recombinant adenovirus vector (E1 minus) expressing human wild-type p53 cDNA (AdWTp53). Infection of normal and tumor cells of lung and mammary epithelial origin with AdWTp53 resulted in high levels of wild-type p53 expression. Production of p53 protein following infection was dependent on the dose of AdWTp53 with maximum amounts of p53 produced following infection with 50 plaque-forming units/cell. AdWTp53 infection inhibited the growth of all human cell lines studied. However, tumor cells that were null for p53 prior to infection (H-358 and MDA-MB-157) and tumor cells that expressed mutant endogenous p53 protein (MDA-MB-231 and MDA-MB-453) were more sensitive to AdWTp53 cytotoxicity than cells that contained the wild-type p53 (MCF-7, MCF-10, 184B5, and normal mammary epithelial cells). All cells exhibited WAF1/Cip1 mRNA and protein induction following AdWTp53 infection. AdWTp53-induced cytotoxicity of human tumor cell lines expressing mutant p53 was mediated by apoptosis as revealed by nucleosomal DNA fragmentation analysis. No detectable nucleosomal DNA fragmentation was observed following AdWTp53 infection of human cells expressing wild-type p53. These data suggest that endogenous p53 status is a determinant of AdWTp53-mediated cell killing of human tumor cells.     

Superior vena cava syndrome caused by chronic hemodialysis catheters: autologous reconstruction with a pericardial tube graft

Although interleukin (IL)-12 was originally purified from an Epstein-Barr (EBV)-transformed B cell line and the high correlation of EBV infection and IL-12 expression has been suggested, no study has reported whether EBV infection is directly linked to IL-12 expression. To address this issue, we have investigated IL-12 expression in B cells during in vitro transformation with EBV. Human peripheral B cells became capable of constitutively producing p40 by in vitro transformation with EBV, coincident with the expression of latent membrane protein 1 (LMP1) of EBV. These B cells expressed p40 and p35 mRNA, and phorbol myristate acetate (PMA) stimulation strongly enhanced p40 and p70 production. Furthermore, transfection with LMP1 expression vector into a human B lymphoma cell line, Daudi, led to p40 production with nuclear factor (NF)-kappaB activation. These results suggest that transformation of primary B cells with EBV induces IL-12 expression potentially through LMP1 expression.     

WAF1/p21 regulates proliferation, but does not mediate p53-dependent apoptosis in urothelial carcinoma cell lines

The WAF1/p21 gene product is an inhibitor of cyclin-dependent kinases which can be induced by the tumor suppressor p53 and mediate some of its effects, or function in p53-independent pathways of cell cycle regulation. Although a potential tumor suppressor gene, WAF1/p21 is expressed in bladder cancer. To elucidate the function of p21 in tumor cells we have investigated in urothelial carcinoma cell lines: i) WAF1/p21 mRNA and protein expression, ii) the biological effects of p21 overexpression or down-regulation and (iii) whether p21 can be induced by p53. WAF1/p21 mRNA levels examined in four cell lines were comparable to bladder mucosa. One cell line, HT1376, failed to express p21 protein due to a frame shift mutation. Overexpression of WAF1/p21 cDNA inhibited clone formation in three cell lines, whereas transfection with antisense WAF1 increased clone sizes and numbers. WAF1 sense clones showed diminished cell proliferation compared to the parental cell line. Apoptosis- induced wild-type p53 was not inhibited by overexpression of antisense WAF1/p21. In a cell clone derived from line VMCub1 by stable transfection with wild-type p53 under the control of a metallothionein promotor, p21 was induced along with p53 upon activation of the promoter with zinc chloride. This induction was accompanied by a decrease in cell proliferation but by little apoptosis. These data suggest that p21 inhibits proliferation in a p53-dependent or independent manner but does not mediate p53-induced apoptosis in urothelial carcinoma cells.     

Survey of nurse anesthesia practice, education, and regulation in 96 countries

Reconstitution of the p53-dependent apoptotic pathway by gene transfer of a recombinant wild-type p53 minigene leads to rapid apoptotic cell death in breast and other cancer cell types expressing null or mutant p53. Tumour cells expressing wild-type p53 have been reported to be more resistant to this treatment strategy, presumably as a result of mutations in downstream regulators of p53-dependent apoptotic signalling. The MCF-7 breast cancer cell line is representative of this class of tumour cell. Our recent observation of a p53-dependent apoptotic response following adenovirus-mediated HSV thymidine kinase gene transfer and gancyclovir treatment led us to reexamine recombinant p53 cytotoxicity in MCF-7 cells. Infection with a recombinant adenovirus expressing wild-type p53 resulted in a dramatic increase in p53 protein levels and was accompanied by an increase in p21WAF/CIP1 protein levels and G1 arrest within 24 hours post-infection. A significant decrease in MCF-7 cell viability was first observed at 5 days post-infection and coincided with the appearance of morphological and biochemical changes consistent with apoptotic cell death. By day 7 post-treatment, cell viability decreased to 45% and clonogenic survival was reduced to 12% of controls. The results demonstrate that persistent, high level expression of recombinant p53 can induce programmed cell death in MCF-7 cells. While the mechanism by which p53 overexpression overcomes the defect in downstream apoptotic signalling is not clear, our data suggests that this treatment strategy may be beneficial for the class of tumour cells represented by the MCF-7 cell line.