Human gene marker/therapy clinical protocols.
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Clinical Protocols↗
Biomedical subjects
J A Roth
Publications and source records attributed to J A Roth.
Down-regulation of bcl-2 is associated with p16INK4-mediated apoptosis in non-small cell lung cancer cells.
We introduced a functional p16 cDNA into non-small cell lung cancer (NSCLC) cell lines expressing different combinations of normal and mutated p16, p53, and Rb genes via a recombinant adenovirus to determine the effect of exogenous p16 expression on cell growth. Analysis of p16-deficient cells infected with Adv/p16 identified growth arrest of the cells in the G0 - G1 phase early on. Apoptosis was identified to occur by the 5th day after infection which corresponded with increased p16 expression, reduced Rb expression, and increased Rb hypophosphorylation, but only occurred in cells expressing functional p53. Further analysis indicated that the expression of the anti-apoptotic protein bcl-2 was greatly reduced in the NSCLC cell lines H460 and A549 (both -p16, +p53, +Rb), again only by the 5th day after Adv/p16 infection, but no affect on Bax expression was observed. H1299 cells (-p16, -p53, +Rb) infected with Adv/p16 only exhibited apoptosis by an additional infection with Adv/p53 which also corresponded with a down-regulation of bcl-2. In addition, the infection of A549 cells with Adv/p16 followed by a subsequent infection with Adv/Rb lead to a significant decrease in apoptosis which correlated with an increase in bcl-2 expression. These studies suggest that p16 is capable of mediating apoptosis in NSCLC cell lines expressing wild-type p53, through a direct down-regulation of Rb and an indirect down-regulation of the anti-apoptotic protein bcl-2.
Adenoviridae↗
Antitumor effect of adenovirus-mediated Bax gene transfer on p53-sensitive and p53-resistant cancer lines.
Antitumor effects of the proapoptotic Bax gene have been evaluated in vitro and in vivo by a binary adenovirus system expressing the human Bax gene. Overexpression of the Bax gene in cultured cell lines from human lung carcinoma results in caspase activation, apoptosis induction, and cell growth suppression. Intratumoral injection of adenovirus vector expressing the Bar gene suppressed growth of human lung cancer xenografts established in nude mice. Histological examination of tumors from mice treated with the Bax gene demonstrated high levels of Bax expression and extensive apoptosis in tumors. In comparison with the treatment by an adenoviral vector expressing human p53, the Bax gene can effectively suppress tumor growth in both p53-sensitive and p53-resistant human lung carcinoma cell lines. Toxicity was not detected in liver and other systems in animals treated intralesionally with the Bax gene. Therefore, our results suggest that the Bar gene may be useful in cancer treatment.
Adenoviridae↗
Adenoviral Bak overexpression mediates caspase-dependent tumor killing.
One of the most promising strategies in cancer gene therapy is adenoviral transfer of proapoptotic genes. We therefore evaluated the novel strategy of adenovirally overexpressing the proapoptotic Bak gene. Our results showed marked apoptosis in cancer cells in vivo and in vitro after Bak gene transfer via a binary adenoviral vector system. This effect was not seen in a caspase 3-defective cell line (MCF-7) and was abrogated in Bak-sensitive tumors after administration of the caspase inhibitor z-DEVD-fmk. Our results suggest that adenoviral-mediated overexpression of Bak provides a novel therapeutic strategy for cancer therapy, but this process appears to be caspase dependent.
Adenoviridae↗
Overexpression of the wild-type p53 gene inhibits NF-kappaB activity and synergizes with aspirin to induce apoptosis in human colon cancer cells.
The tumor suppressor gene p53 is a potent transcriptional regulator of genes which are involved in many cellular activities including cell cycle arrest, apoptosis, and angiogenesis. Recent studies have demonstrated that the activation of the transcriptional factor nuclear factor kappaB (NF-kappaB) plays an essential role in preventing apoptotic cell death. In this study, to better understand the mechanism responsible for the p53-mediated apoptosis, the effect of wild-type p53 (wt-p53) gene transfer on nuclear expression of NF-kappaB was determined in human colon cancer cell lines. A Western blot analysis of nuclear extracts demonstrated that NF-kappaB protein levels in the nuclei were suppressed by the transient expression of the wt-p53 in a dose-dependent manner. Transduced wt-p53 expression increased the cytoplasmic expression of I kappaB alpha as well as its binding ability to NF-kappaB, thus markedly reducing the amount of NF-kappaB that translocated to the nucleus. The decrease in nuclear NF-kappaB protein correlated with the decreased NF-kappaB constitutive activity measured by electrophoretic mobility shift assay. Furthermore, parental cells transfected with NF-kappaB were better protected from cell death induced by the wt-p53 gene transfer. We also found that the wt-p53 gene transfer was synergistic with aspirin (acetylsalicylic acid) in inhibiting NF-kappaB constitutive activity, resulting in enhanced apoptotic cell death. These results suggest that the inhibition of NF-kappaB activity is a plausible mechanism for apoptosis induced by the wt-p53 gene transfer in human colon cancer cells and that anti-NF-kappaB reagent aspirin could make these cells more susceptible to apoptosis.
Adenocarcinoma↗
Molecular events in bronchogenic carcinoma and their implications for therapy.
Lung cancer patients suffer a 15% overall survival despite advances in chemotherapy, radiation therapy and surgery. This unacceptably low survival rate is due to the usual finding of advanced disease at diagnosis. Detecting preneoplastic lesions and determining which invasive lesions are prone to metastasize or recur can be formidable tasks. Understanding the molecular events that occur during the multistep process of bronchogenic carcinogenesis may make these tasks more surmountable. Approaches leading to identification of patients susceptible to cancer formation include detection of poor metabolizers of carcinogens, those unable to repair genetic alterations, those with activated oncogenes or inactivated tumor suppressor genes, and those at risk of poor outcomes. Detection may be achieved at the cellular, chromosomal, genetic, or protein level. Novel therapies can then be developed that prevent tumor initiation into and promotion through the multistep carcinogenesis pathway, conversion from preneoplastic into invasive malignancies, and progression into metastasis or recurrences. Therapeutic success of chemoprevention can be followed by changes in molecular marker levels. Blockade of activated tumor-promoting oncogenes or replacement of inactivated tumor-suppressing or apoptosis-promoting genes can be achieved by gene therapy. Development of these novel molecular diagnostic and therapeutic strategies could result in prevention of cancer formation or at least prolongation of disease-free survival.
Carcinoma, Bronchogenic↗
Gene therapy in lung cancer.
The poor overall survival rates associated with non- small-cell lung cancer despite advances in chemotherapy, radiotherapy, and surgery mandate the search for novel approaches. Advances in gene transfer technology have allowed gene therapy strategies to develop that act in such a way as to stimulate the immune system, transfer "suicide" genes, inactivate oncogenes, replace tumor suppressor genes, and transfer pro-apoptotic genes. Clinical trials evaluating these possibilities have begun, and findings indicate that the transfer of tumor sup- pressor genes (wild-type p53) is feasible and has low overall toxicity. Subsequent clinical trials have begun to evaluate the clinical potential of these approaches in non-small-cell lung cancer.
Apoptosis↗
Outpatient management of malignant pleural effusion by a chronic indwelling pleural catheter.
BACKGROUND: Previous studies have shown that a chronic indwelling pleural catheter (PC) safely and effectively relieved dyspnea, maintained quality of life, and reduced hospitalization in patients with malignant pleural effusions. Outpatient management of malignant pleural effusion with a PC may reduce length of stay and early (7-day) charges compared with inpatient management with chest tube and sclerosis. METHODS: A retrospective review of consecutive PC patients (n = 100; 60 outpatient, 40 inpatient) were treated from July 1, 1994 to September 2, 1998 and compared with 68 consecutive inpatients treated with chest tube and sclerosis between January 1, 1994 and December 31, 1997. Hospital charges were obtained from date of insertion (day 0) through day 7. RESULTS: Demographics were similar in both groups. Pretreatment cytology was positive in 126 of 168 patients (75%), negative in 21 (12.5%), and unknown in 21 (12.5%). Primary histology included lung (n = 61, 36%), breast (n = 39, 23%), lymphoma (n = 12, 7%), or other (n = 56, 34%). Median survival was 3.4 months and did not differ significantly between treatment groups. Overall median length of stay was 7.0 days for inpatient chest tube and inpatient PC versus 0.0 days for outpatient Pleurx. No mortality occurred related to the PC. Eighty-one percent (81/100) of PC patients had no complications. One or more complications occurred in 19 patients (19%). Patients treated with outpatient PC (n = 60) had early (7-day) mean charges of $3,391 +/- $1,753 compared with inpatient PC (n = 40, $11,188 +/- $7,964) or inpatient chest tube (n = 68, $7,830 +/- $4,497, SD) (p < 0.001). CONCLUSIONS: Outpatient PC may be used effectively and safely to treat malignant pleural effusions. Hospitalization is not required in selected patients. Early (7-day) charges for malignant pleural effusion are reduced in outpatient PC patients compared with inpatient PC patients or chest tube plus sclerosis patients.
Aged↗
Cellular and humoral immune responses to adenovirus and p53 protein antigens in patients following intratumoral injection of an adenovirus vector expressing wild-type. P53 (Ad-p53).
The immune responses of 10 patients with advanced non-small cell lung cancer receiving monthly intratumoral injections of a recombinant adenovirus containing human wild-type p53 (Ad-p53) to adenovirus and transgene antigens were studied. The predominate cellular and humoral immune responses as measured by lymphocyte proliferation and neutralizing antibody (Ab) formation were to adenovirus serotype 5 vector antigens, with increased responses in posttreatment samples. Consistent alterations in posttreatment cellular and humoral immune responses to p53 epitopes were not observed, and cytotoxic Abs to human lung cancer cells were not generated. Patients in this study had evidence of an antitumoral effect of this treatment with prolonged tumor stability or regression; however, neither Abs to p53 protein nor increased lymphocyte proliferative responses to wild-type or mutant p53 peptides have been consistently detected.
Adenoviridae↗
Synergistic effects of adenovirus expressing wild-type p53 on chemosensitivity of non-small cell lung cancer cells.
The infection of recombinant adenovirus expressing wild-type p53 (Ad-p53) to lung cancer cells that harbor mutant p53 genes improves their response to cis-diamminedichloroplatinum(II). In this study, we tested whether this improvement in response is also seen in wild-type p53 (wt-p53)-containing cancer cells and whether this phenomenon is universal with other commonly used chemotherapeutic agents, including etoposide, 7-ethyl-10-hydrocycamptothecin, paclitaxel, and docetaxel. Using a panel of 7 non-small cell lung cancer cell lines with wild-type (2) or abnormal (2 null, 3 point-mutated) p53, we examined in vitro cytotoxicity using a tetrazolium-based colorimetric assay (3-(4,5-diethylthiazoyl-2-yl)-2,5-diphenyltetrazolium bromide assay) and analyzed the combined effects of Ad-p53 and chemotherapeutic agents using the isobologram method. Ad-p53 and DNA-damaging agents (cis-diamminedichloroplatinum(II), etoposide, and 7-ethyl-10-hydrocycamptothecin) showed synergistic effects in six of seven cell lines but additive effects against a p53-mutated cell line. In contrast, Ad-p53 showed additive effects with the antitubulin agents (paclitaxel and docetaxel) in all four of the cell lines tested. Furthermore, we examined this synergistic interaction between Ad-p53 and DNA-damaging agents by flow cytometric analysis and DNA fragmentation analysis. Both analyses revealed that a sublethal dose of Ad-p53 augmented the apoptotic response induced by DNA-damaging agents in six of seven cell lines. Our results suggest that Ad-p53 may synergistically enhance the chemosensitivity of the majority of non-small cell lung cancers to DNA-damaging agents due to augmentation of apoptosis.
Adenoviridae↗
A binary adenoviral vector system for expressing high levels of the proapoptotic gene bax.
The bax gene plays a critical role in signaling apoptosis and expression through gene transfer may be valuable in the treatment of a variety of apoptosis-related diseases such as cancer. However, constructing an adenoviral vector expressing a bax gene driven by a constitutive promoter has been difficult, presumably because of the gene's high proapoptotic activity. Here we report a system that induces the expression of the bax gene safely by adenovirus-mediated gene cotransfer. Briefly, the system involves an adenoviral vector containing a human bax cDNA driven by a synthetic promoter consisting of five GAL4-binding sites and a TATA box (GT). This vector expresses a minimal background level of bax protein in cultured mammalian cells thus preventing apoptosis of packaging cells, however, expression of the bax gene can be induced substantially in vitro and in vivo by transferring it into target cells along with an adenoviral vector expressing the transactivator, fusion protein GAL4/VP16. Extensive apoptosis was observed after induction of the bax gene both in cultured human lung carcinoma cells and in the livers of Balb/c mice. Our results suggest that this GAL4 gene regulatory system provides an alternative approach to constructing viral vectors that express potentially toxic genes. Gene Therapy (2000) 7, 75-79.
Adenoviridae↗
Tumor-suppressive effects by adenovirus-mediated mda-7 gene transfer in non-small cell lung cancer cell in vitro.
The melanoma differentiation-associated gene-7 (mda-7), cloned from a human melanoma cell line H0-1, is known to induce tumor cell-selective growth inhibition in breast cancer cells in vitro and loss of tumorigenicity ex vivo. Yet, the mechanisms underlying these effects are still unknown. Therefore, we investigated these mechanisms on the molecular level in human non-small cell lung carcinoma (NSCLC) cells in vitro. Overexpression of mda-7 protein by Ad-mda-7 significantly suppressed proliferation and induced G2/M cell cycle arrest in wild-type p53 (A549, H460), and p53-null (H1299) non-small cell lung cancer cell lines, but not in normal human lung fibroblast (NHLF) cells. p53, Bax, and Bak protein expression was up-regulated in wild-type p53 tumor cell lines, but not in p53-null cells, suggesting that an intact p53 pathway was required for Bax and Bak induction. However, in all three cancer cell lines tested, activation of the caspase cascade and cleavage of poly(ADP-ribose) polymerase (PARP) appeared to be independent of the p53 mutational status. Together, these results suggest that apoptosis may be induced via multiple pathways by Ad-mda-7 in lung cancer cells and that Ad-mda-7 has the potential to become a novel therapeutic for clinical cancer gene therapy. Gene Therapy (2000) 7, 2051-2057.
Adenoviridae↗
Effects of danofloxacin and tilmicosin on circulating neutrophils in beef heifers.
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Animals↗
Gene replacement strategies for treating non-small cell lung cancer.
Fewer than 15% of the 170,000 patients who develop lung cancer each year will survive their disease, which shows the need for novel, more specific, and less toxic therapeutic strategies. Recent advances in molecular biology have made it possible to ascertain which genetic alterations contribute to the etiology of cancer. For example, the tumor-suppressor gene, p53, responsible for directing repair of damaged DNA or committing a cell to apoptosis, is mutated or otherwise altered in more than 50% of cancers, including 40% to 70% of non-small cell lung cancers. Many p53-deficient tumors have proven remarkably resistant to radiotherapy and chemotherapy. The preclinical and clinical studies of gene therapy reviewed in this article show (1) successful transfer and expression of a potentially therapeutic p53 gene construct in tumor cells, (2) observation of antitumor effects in vitro and in vivo, and (3) most critically, a lack of significant toxicity. The results of these studies indicate that gene replacement therapy is a feasible alternative therapy for cancer. In addition, these studies show that transfer of the p53 gene can induce radiation sensitization in previously radiation-resistant tumors, leading to the possibility of new therapeutic protocols combining gene replacement with radiation therapy.
Apoptosis↗
Multiple molecular marker testing (p53, C-Ki-ras, c-erbB-2) improves estimation of prognosis in potentially curative resected non-small cell lung cancer.
A prospective study was performed in patients with non-small cell lung cancer (NSCLC) to evaluate the prognostic importance of multiple molecular marker (p53, c-Ki-ras, c-erbB-2) testing. 103 patients with potentially curative resections (RO resection) for NSCLC in histopathological stages I-IIIA were included. SSCP analysis and DNA sequencing for p53 and c-Ki-ras genes were performed on paired tumour and normal lung tissue samples and immunohistochemistry (c-erbB-2) was done on frozen tissue sections with a specific anti-c-erbB-2 monoclonal antibody. 46/103 (44.6%) NSCLC showed p53 mutations and 17/103 (16.5%) c-Ki-ras mutations including 12/37 (32.4%) adenocarcinomas. Overexpression of c-erbB-2 (p185) was detected in 56/103 (54.4%) tumours. 24/103 (23.3%) NSCLC were negative for alterations in all 3 parameters (c-Ki-ras, p53 and p185) whereas 79/103 (76.7%) were positive for at least one of the 3 parameters. In a regression model including a multiple molecular marker parameter (negative for all 3 markers versus positive for at least one marker), histopathological stage (P<0.00001), respectively the pT (P<0.01) and pN (P<0.00001) categories and the multiple molecular marker parameter (P<0.01) were of significant prognostic importance. This study demonstrates that testing 3 molecular markers (c-Ki-ras, p53 and c-erbB-2) improves estimation of prognosis compared to single marker testing and appears to define low (82.6%+/-7.9% 5-year survival) and high risk (40.2%+/-5.5% 5-year survival) groups for treatment failure in potentially curative (RO) resected NSCLC.
Adult↗
Effect of operative volume on morbidity, mortality, and hospital use after esophagectomy for cancer.
OBJECTIVE: We sought to evaluate the effect of operative volume, hospital size, and cancer specialization on morbidity, mortality, and hospital use after esophagectomy for cancer. METHODS: Data derived from the Health Care Utilization Project was used to evaluate all Medicare-reimbursed esophagectomies for treatment of cancer from 1994 to 1996 in 13 national cancer institutions and 88 community hospitals. The complications of care, length of stay, hospital charges, and mortality were assessed according to hospital size (>/=600 beds vs <600 beds), cancer specialization (national cancer institution vs community hospital), and operative volume (esophageal [>/=5 Medicare esophagectomies per year vs <5 Medicare esophagectomies per year] and nonesophageal operations [>/=3333 cases per year vs <3333 cases per year]). RESULTS: Mortality was lower in national cancer institution hospitals (4.2% [confidence interval, 2.0%-6.4%] vs 13.3% [confidence interval, 4.2%-26.2%], P =. 05) and in hospitals performing a large number of esophagectomies (3. 0% [confidence interval, 0.09%-5.1%] vs 12.2% [confidence interval, 4.5%-19.8%], P <.05). Multivariate analysis revealed that the independent risk factor for operative mortality was the volume of esophagectomies performed (odds ratio, 3.97; P =.03) and not the number of nonesophageal operations, hospital size, or cancer specialization. Hospitals performing a large number of esophagectomies also showed a tendency toward decreased complications (55% vs 68%, P =.06), decreased length of stay (14.7 days vs 17.7 days, P =.006), and decreased charges ($39,867 vs $62, 094, P <.005). CONCLUSIONS: These results demonstrate improved outcomes and decreased hospital use in hospitals that perform a large number of esophagectomies and support the concept of tertiary referral centers for such complex oncologic procedures as esophagectomies.
Aged↗
Antigenic and genetic stability of bovine immunodeficiency virus during long-term persistence in cattle experimentally infected with the BIV(R29) isolate.
Experimental infection of cattle with bovine immunodeficiency virus (BIV) is characterized by persistent, low levels of virus replication in the absence of clinical disease. A virus neutralization (VN) assay was developed to examine the role of VN antibodies in controlling virus replication in cattle experimentally infected with the BIV(R29) isolate of BIV. All animals developed VN antibody, but there was no correlation between VN titres and restriction of virus replication in vivo. BIV infection did not induce high-titred, cross-neutralizing antibody and there was no evidence for antigenic variation through more than 4 years in vivo. Genetic comparisons among the BIV(R29) inoculum virus and viruses isolated from infected animals identified only limited genetic variation during 4 years in vivo. Moreover, there was no evidence that the observed variation was due to selection. Analyses of genetic diversity in the virus stock used for inoculation indicated a fairly homogeneous population. In the absence of high levels of virus replication and overt clinical disease, there appeared to be little selection of virus variants, resulting in antigenic and genetic stability of BIV(R29) during long-term, persistent infection.
Amino Acid Sequence↗
Multiple lysine mutations in the C-terminal domain of p53 interfere with MDM2-dependent protein degradation and ubiquitination.
To investigate the effect of mutations in the p53 C-terminal domain on MDM2-mediated degradation, we introduced single and multiple point mutations into a human p53 cDNA at four putative acetylation sites (amino acid residues 372, 373, 381, and 382). Substitution of all four lysine residues by alanines (the A4 mutant) and single lysine-to-alanine substitutions were functional in sequence-specific DNA binding and transactivation; however, the A4 mutant protein was resistant to MDM2-mediated degradation, whereas the single lysine substitutions were not. Although the A4 mutant protein and the single lysine substitutions both bound MDM2 reasonably well, the single lysine substitutions underwent normal MDM2-dependent ubiquitination, whereas the A4 protein was inefficiently ubiquitinated. In addition, the A4 mutant protein was found in the cytoplasm as well as in the nucleus of a subpopulation of cells, unlike wild-type p53, which is mostly nuclear. The partially cytoplasmic distribution of A4 mutant protein was not due to a defect in nuclear import because inhibition of nuclear export by leptomycin B resulted in nuclear accumulation of the protein. Taken together, the data suggest that mutations in the putative acetylation sites of the p53 C-terminal domain interfere with ubiquitination, thereby regulating p53 degradation.
Acetylation↗