Biomedical subjects

David J McConkey

Publications and source records attributed to David J McConkey.

49 records · Page 3Linked to original sources

PubMed2002

Development of biologic markers of response and assessment of antiangiogenic activity in a clinical trial of human recombinant endostatin.

PURPOSE: Angiogenesis is a target for the treatment of cancer and other diseases, and its complex biology suggests that establishing the appropriate dose and schedule for antiangiogenic treatment will require extensive study. We present the initial results of a dose-finding clinical trial of recombinant human endostatin (rh-Endo) that examined potential surrogates for response to antiangiogenic therapy. PATIENTS AND METHODS: Twenty-five patients were treated with escalating doses of rh-Endo. Positron emission tomography (PET) was used to assess tumor blood flow (with [15O]H2O) and metabolism (with [18F]fluorodeoxyglucose) before the start of therapy and then every 4 weeks. To directly assess the effects of rh-Endo on endothelial cells within the tumors, biopsy specimens of tumor tissue were obtained before therapy and again at 8 weeks and evaluated for endothelial cell and tumor cell apoptosis. RESULTS: Tumor blood flow and metabolism as measured by PET scans generally decreased with increasing doses of rh-Endo; however, the effects were complex and in some analyses nonlinear. Tumor biopsy analysis revealed a significant increase in tumor cell apoptosis (P =.027) and endothelial cell apoptosis (P =.027) after 8 weeks of therapy. However, there was no statistically significant relationship between rh-Endo dose and induction of tumor cell or endothelial cell apoptosis. CONCLUSION: These initial data suggest that rh-Endo has measurable effects on tumor blood flow and metabolism and induces endothelial and tumor cell apoptosis even in the absence of demonstrable anticancer effects. Further study and validation of these biomarkers in the context of antiangiogenic therapy will be required.

Adenocarcinoma↗

PubMed2002

Phase I study of recombinant human endostatin in patients with advanced solid tumors.

PURPOSE: Endostatin, a 20-kd fragment of collagen XVIII, is a potent inhibitor of angiogenesis. We evaluated recombinant human endostatin (rh-Endo) in a phase I trial designed to assess safety, pharmacokinetics, and serum markers of angiogenesis in patients with solid tumors. PATIENTS AND METHODS: Twenty-six patients were enrolled onto a dose-finding trial of rh-Endo administered as an intravenous bolus over a 20-minute period once daily. Three patients each were treated at dose levels of 15, 30, 60, 120, 180, and 600 mg/m(2)/d, and seven patients were treated at 300 mg/m(2)/d. Treatment consisted of a minimum of two 28-day cycles. Evaluations included noninvasive imaging, pharmacokinetics, and serum biomarkers. RESULTS: Twenty-five patients were treated with rh-Endo. Treatment was well tolerated; there were no dose-limiting toxic effects. Bacteremia from frequent central line access was the most common problem. The pharmacokinetic disposition of rh-Endo was linear and best described using a two-compartmental open model. The overall mean half-life was 10.7 +/- 4.1 hours. A dose of 300 mg/m(2) achieved an area under the concentration-time curve associated with activity in preclinical models. In two patients, there was evidence of antitumor activity, but no responses were seen. Serum markers of angiogenic activity did not provide insight into rh-Endo's activity. Serum antibodies were observed against both rh-Endo and the Pichia pastoris vector, but no allergic reactions were observed. CONCLUSION: rh-Endo was safe and well tolerated. rh-Endo pharmacokinetic profiles achieved area under the concentration-time curves associated with activity in preclinical models. Evidence of minor antitumor activity was observed and further studies are indicated.

Adenocarcinoma↗

PubMed2002

Role of P38 MAPK, AP-1, and NF-kappaB in interleukin-1beta-induced IL-8 expression in human vascular smooth muscle cells.

Interleukin (IL)-1 modulates the expression of various genes in normal and tumor cells. We investigated the molecular mechanisms underlying IL-1beta-induced expression of IL-8 mRNA and protein in human vascular smooth muscle cells (hVSMCs). P38 mitogen-activated protein kinase (MAPK) was activated after 5min of IL-1beta treatment, whereas the extracellular signal-regulated kinases, the c-jun amino-terminal kinases, and protein kinase B/Akt were not activated by IL-1beta. IL-1beta induced activation of a full-length IL-8 promoter-reporter construct. Deletional mutagenesis localized the IL-1beta-responsive domains to two regions (-133 to -98 and -85 to -50) that contain consensus binding sites for activator protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB), respectively. Site-directed mutagenesis of the 133-bp minimal promoter confirmed that these sites were required for promoter activity. Electrophoretic mobility shift assays confirmed that IL-1beta increased AP-1 and NF-kappaB DNA-binding activities in a time-dependent manner. SB203580, a specific P38 MAPK inhibitor, partially blocked IL-1beta induction of IL-8 mRNA, IL-8 promoter activity, and AP-1 nuclear extract binding but not NF-kappaB DNA binding. Our data demonstrate that AP-1 and NF-kappaB are essential transcription factors for IL-1beta-induced IL-8 gene expression in hVSMCs. P38 MAPK is involved in inducing IL-8 gene transcription via AP-1 activation in hVSMCs.

Binding Sites↗

PubMed2002

Anoikis is regulated by BCL-2-independent pathways in human prostate carcinoma cells.

BACKGROUND: Loss of contact with the extracellular matrix (ECM) triggers a specialized form of apoptosis known as "anoikis" in normal epithelial cells. Dependence on adhesion to ECM is often lost in transformed cells, and the degree of anchorage independence may vary in non-metastatic and metastatic cancer cells. BCL-2 oncoprotein overexpression correlates with the progression and metastases of prostate cancer. Materials and Methods We studied anoikis in suspension cultures of PC-3 and LNCaP prostate carcinoma cells selected for enhanced metastatic potential in vivo and in PC-3 and LNCaP cells stably transfected with BCL-2. Apoptosis-associated DNA fragmentation was measured by agarose gel electrophoresis and propidium iodide staining and flow cytometry. Expression of BCL-2 family polypeptides was determined by immunoblotting. RESULTS: Non-metastatic PC-3P cells were significantly more sensitive to anoikis than the metastatic PC-3 variants (PC-3M, PC-3M-PRO-4, and PC-3M-LN-4), but anoikis resistance did not correlate with metastatic potential in LNCaP-derived cell lines. Expression of BCL-2 was higher in metastatic PC-3 and LNCaP subclones compared to isogenic non-metastatic cells, but these levels were not affected by anoikis. Enforced overexpression of BCL-2 did not protect either PC-3P or LNCaP-PRO-5 cells from anoikis, even though it rendered them resistant to thapsigargin and inhibited cytochrome c release. Strikingly, cells that died of anoikis maintained their pretreatment levels of BCL-2, whereas the cells that survived anoikis expressed much lower levels of the protein. CONCLUSIONS: Sensitivity to anoikis is regulated by BCL-2 independent mechanisms in LNCaP and PC-3 prostate cancer cells.

Adenocarcinoma↗

PubMed2002

Bax-mediated Ca2+ mobilization promotes cytochrome c release during apoptosis.

Previous studies have demonstrated that Ca(2+) is released from the endoplasmic reticulum (ER) in some models of apoptosis, but the mechanisms involved and the functional significance remain obscure. We confirmed that apoptosis induced by some (but not all) proapoptotic stimuli was associated with caspase-independent, BCL-2-sensitive emptying of the ER Ca(2+) pool in human PC-3 prostate cancer cells. This mobilization of ER Ca(2+) was associated with a concomitant increase in mitochondrial Ca(2+) levels, and neither ER Ca(2+) mobilization nor mitochondrial Ca(2+) uptake occurred in Bax-null DU-145 cells. Importantly, restoration of DU-145 Bax expression via adenoviral gene transfer restored ER Ca(2+) release and mitochondrial Ca(2+) uptake and dramatically accelerated the kinetics of staurosporine-induced cytochrome c release, demonstrating a requirement for Bax expression in this model system. In addition, an inhibitor of the mitochondrial Ca(2+) uniporter (RU-360) attenuated mitochondrial Ca(2+) uptake, cytochrome c release, and DNA fragmentation, directly implicating the mitochondrial Ca(2+) changes in cell death. Together, our data demonstrate that Bax-mediated alterations in ER and mitochondrial Ca(2+) levels serve as important upstream signals for cytochrome c release in some examples of apoptosis.

Apoptosis↗

PubMed2002

Disruption of Mekk2 in mice reveals an unexpected role for MEKK2 in modulating T-cell receptor signal transduction.

MEKK2 is a member of the mitogen-activated protein kinase (MAPK) kinase kinase gene family involved in regulating multiple MAPK signaling pathways. To elucidate the in vivo function of MEKK2, we generated mice carrying a targeted mutation in the Mekk2 locus. Mekk2(-/-) mice are viable and fertile. Major subsets of thymic and spleen T cells in Mekk2-deficient mice were indistinguishable from those in wild-type mice. B-cell development appeared to proceed similarly in the bone marrow of Mekk2-deficient and wild-type mice. However, Mekk2(-/-) T-cell proliferation was augmented in response to anti-CD3 monoclonal antibody (MAb) stimulation, and these T cells produced more interleukin 2 and gamma interferon than did the wild-type T cells, suggesting that MEKK2 may be involved in controlling the strength of T-cell receptor (TCR) signaling. Consistently, Mekk2(-/-) thymocytes were more susceptible than wild-type thymocytes to anti-CD3 MAb-induced cell death. Furthermore, TCR-mediated c-Jun N-terminal kinase activation was not blocked but moderately enhanced in Mekk2(-/-) T cells. Neither extracellular signal-regulated kinase nor p38 MAPK activation was affected in Mekk2(-/-) T cells. In conclusion, we found that MEKK2 may be required for controlling the strength of TCR/CD3 signaling.

Animals↗

PubMed2002

Inhibition of the phosphatidylinositol 3'-kinase-AKT pathway induces apoptosis in pancreatic carcinoma cells in vitro and in vivo.

The phosphatidylinositol 3'-kinase (PI3k)-AKT survival pathway is activated in many malignancies. We observed constitutive AKT phosphorylation (on S473) consistent with pathway activation in seven of nine human pancreatic carcinoma cell lines in vitro. Exposure of the cells to two structurally distinct inhibitors of PI3k (worthmannin and LY294002) resulted in a dose-dependent induction of apoptosis in six of seven of the cell lines that displayed constitutive AKT phosphorylation but not in either of the cell lines that did not. The mitogen-activated protein/extracellular signal-regulated kinase kinase-mitogen-activated protein kinase inhibitor PD98059 also induced apoptosis in two of the cell lines, including one of the LY294002-insensitive lines (AsPC-1). Exposure of orthotopic L3.6pl pancreatic tumors to LY294002 resulted in dose-dependent inhibition of tumor growth, and decreased peritoneal and liver metastases, effects that were associated with an inhibition of AKT phosphorylation and increased terminal deoxynucleotidyl transferase-mediated nick end labeling staining characteristic of apoptosis. Furthermore, a suboptimal dose of LY294002 (25 mg/kg) produced additive inhibition of tumor growth when combined with a suboptimal dose of gemcitabine (62 mg/kg). Together, our results establish that the PI3k/AKT pathway is constitutively activated in a majority of human pancreatic cancer cell lines and establish that the pathway is a promising target for therapeutic intervention.

Androstadienes↗

PubMed2002

Effects of the proteasome inhibitor PS-341 on apoptosis and angiogenesis in orthotopic human pancreatic tumor xenografts.

Recent studies have shown that the transcription factor, nuclear factor kappaB (NF-kappaB), regulates critical survival pathways in a variety of different cell types, including human pancreatic cancer cells. The activation of NF-kappaB is controlled by proteasome-mediated degradation of its endogenous polypeptide inhibitor, inhibitor of nuclear factor kappaBalpha. We investigated the effects of PS-341, a peptide boronate inhibitor of the proteasome in human pancreatic cancer cells in vitro and in vivo. Comparison of PS-341's effects on the growth of eight different human pancreatic cancer cell lines revealed marked heterogeneity in drug responsiveness, ranging from highly resistant (IC50 > 10 microM; Panc-48, HS766T, and Mia-PaCa-2) to extremely sensitive (IC50 < 40 nM; L3.6pl, Hpaf2, and BxPC3). However, these effects did not correlate with differential inhibition of NF-kappaB activation. Direct quantification of apoptosis revealed that PS-341's effects on cell growth largely correlated with sensitivity to programmed cell death. Evaluation of PS-341's effects on established orthotopic tumor xenografts demonstrated that biweekly intravenous administration of the maximum-tolerated dose of the drug (1 mg/kg) led to significant reductions in the volumes of L3.6pl tumors but not Mia-PaCa-2 tumors. Laser scanning cytometer-mediated quantification of drug-induced apoptosis in the xenografts confirmed that PS-341 induced DNA fragmentation and activation of caspase-3 in L3.6pl tumors but not in Mia-PaCa-2 tumors. However, histological examination of drug-treated tumors revealed extensive central necrosis and reductions in microvessel density and VEGF expression in both tumor types. Taken together, our results demonstrate that PS-341 inhibits the growth of human pancreatic tumors via direct effects on tumor cells and indirect effects on the tumor vasculature.

Animals↗

PubMed2002

Adenoviral transfer of mda-7 leads to BAX up-regulation and apoptosis in mesothelioma cells, and is abrogated by over-expression of BCL-XL.

BACKGROUND: Malignant pleural mesothelioma (MPM) is unresponsive to conventional therapies. Forced expression of the novel tumor suppressor mda-7 gene in other cell types has resulted in decreased growth and apoptosis. We evaluated cell growth, apoptosis and tumor suppressor characteristics following forced expression of this gene in mesothelioma cell lines. METHODS: MDA-7 expression in human MPM cells at baseline, following pharmacologic differentiation and viral mda-7 transduction (Ad-mda7) were evaluated with Western blot. Cell viability was evaluated with a colorimetric (XTT) assay, and apoptosis with subG1 FACS and Hoescht. Caspase-3 expression was evaluated by functional assay. These parameters were also evaluated in a stable bcl-xl hyper-expressing MPM cell line. Bax mRNA levels were evaluated with real-time PCR. RESULTS: No baseline or differentiated MPM MDA7 expression was found, but was noted following Ad-mda7 exposure. More than 50% of MPM cells were killed at 5 days following Ad-mda7 exposure (p < 0.001). Apoptosis was accompanied by caspase-3 cleavage and increased BAX expression at both the protein (translational) and mRNA (transcriptional) level. These findings were reduced in a bcl-xl hyper-expressing cell line (P < 0.01). CONCLUSIONS: Although mda-7 does not appear to be a MPM suppressor gene, adenoviral-mediated expression in cell lines induces apoptotic cellular death related to BAX upregulation and caspase cleavage. This is supported by abrogation of effect in a bcl-xl hyper-expressing cell line.

Adenoviridae↗

PubMed2001

Bax and Bak promote apoptosis by modulating endoplasmic reticular and mitochondrial Ca2+ stores.

Alterations in intracellular Ca(2+) homeostasis and cytochrome c release from mitochondria have been implicated in the regulation of apoptosis, but the relationship between these events remains unclear. Here we report that enforced expression of either Bax or Bak via adenoviral gene delivery results in the accumulation of the proteins in the endoplasmic reticulum (ER) and mitochondria, resulting in early caspase-independent BCL-2-sensitive release of the ER Ca(2+) pool and subsequent Ca(2+) accumulation in mitochondria. The inhibition of ER-to-mitochondrial Ca(2+) transport with a specific inhibitor of mitochondrial Ca(2+) uptake attenuates cytochrome c release and downstream biochemical events associated with apoptosis. Bax and Bak also directly sensitize mitochondria to cytochrome c release induced by immediate emptying of ER Ca(2+) pool. Our results demonstrate that the effects of the "multidomain" proapoptotic BCL-2 family members Bak and Bax involve direct effects on the endoplasmic reticular Ca(2+) pool with subsequent sensitization of mitochondria to calcium-mediated fluxes and cytochrome c release. These effects modulate the kinetics of cytochrome c release and apoptosis.

Apoptosis↗

PubMedDate not supplied

Chemotherapy-induced apoptosis and Bcl-2 levels correlate with breast cancer response to chemotherapy.

PURPOSE: The relevance of apoptosis to breast cancer response to chemotherapy is unclear. We investigated whether changes in tumor cell apoptosis and Bcl-2 expression immediately after chemotherapy correlated with response to breast cancer treatment. PATIENTS AND METHODS: Serial core biopsies of 25 breast cancer primary tumors were performed at either two or three time points: before treatment (N = 24) and approximately 24 hours (N = 22) and/or 48 hours (N = 19) after the initiation of the first cycle of chemotherapy. Apoptosis levels were quantified by use of a fluorescent terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) stain, and Bcl-2 and Bax were measured by semiquantitative immunohistochemical assays. All calculated P values were two sided. RESULTS: The apoptosis level at 48 hours was significantly higher in the tumors with pathological complete response or < 1 cm of residual disease (median, 22%; range, 6%-51%) than in the tumors with > 1 cm residual disease (median, 7%; range, 1%-36%); Mann-Whitney test. This difference was also present in the subgroup of 16 tumors treated with docetaxel/doxorubicin chemotherapy (25% vs 4%, respectively). A decrease in Bcl2 expression after chemotherapy relative to the expression from the pretreatment sample also correlated with disease response. Specifically, three of the nine tumors with a decrease in Bcl-2 had a pathological complete response, compared with 0 of the 15 tumors with stable levels of Bcl-2 (Fisher's exact test). There was no relationship between serial measurements of Bax and response. DISCUSSION: These data suggest that apoptosis may play an important role in determining breast cancer response to chemotherapy and that the level of treatment-induced apoptosis may have some value as a predictive marker.

Adult↗

PubMedDate not supplied

Increased apoptosis in metastatic human colonic adenocarcinomas.

Recent work suggests that apoptosis is disrupted during the progression of many solid tumors. Isogenic metastatic colon adenocarcinoma cells displayed significantly higher levels of staurosporine-induced apoptosis compared to their nonmetastatic counterparts in vitro. In addition, analysis of 15 matched primary tumors and liver metastases demonstrated that the levels of apoptosis were significantly higher in the metastases, and this increased cell death was associated with significantly lower levels of Bcl-2 protein expression. Our data demonstrate that the molecular events associated with acquisition of the metastatic phenotype sensitize colon cancer cells to some pro-apoptotic stimuli.

Adenocarcinoma↗

PubMedDate not supplied

Comparative study of various biological parameters, including expression of survivin, between primary and metastatic human colonic adenocarcinomas.

BACKGROUND: Hepatic metastasis of colorectal carcinomas is the most important factor in prognosis. We examined the level of apoptosis and proliferation, the expression of survivin, bcl-2, p53 and intratumoral microvessel density (IMVD) in paired tissue specimens of primary human colon tumors and hepatic metastases and determined the molecular biological changes of the tumor cells in liver metastasis. MATERIALS AND METHODS: We examined, immunohistochemically, the level of apoptosis and proliferation, the expression of survivin, bcl-2 p53 and intratumoral microvessel density in 37 paired tissue specimens of primary colon tumors and hepatic metastases. RESULTS: The mean apoptotic index (AI) was 0.60+/-0.45 for the primary colon tumors and 1.22+/-0.73 for the hepatic metastases. The mean proliferative index (PI) was 37.4+/-15.8 for the primary colon tumors and 29.4+/-14.1 for the hepatic metastases. Significantly higher AI and lower PI were observed in the hepatic metastases as compared to the primary colon tumor (p<0.0001 and p=0.0049, respectively). The mean-weighted survivin score was 4.32+/-2.78 for primary colon carcinomas, and 1.54+/-1.77 for the hepatic metastases. The mean-weighted bcl-2 score was 2.62+/-2.62 for the primary colon carcinomas and 0.81+/-1.56 for the hepatic metastases. There were significantly decreased scores of immunostaining for both survivin and bcl-2 in the hepatic metastases as compared to the primary carcinomas (p<0.0001 and p<0.0002, respectively). Nuclear accumulation of p53 was demonstrated in 25 cases (67.6%) of the primary carcinomas and 24.cases (64.9%) of the hepatic metastases, without significant differences. The IMVD was 89.9+/-37.5 for primary colon tumors, while it was 55.1+/-32.5 for hepatic metastases. A statistically significant decrease in the IMVD was observed in the hepatic metastases as compared to the primary colon tumors (p<0.0001). CONCLUSION: These data suggest that the tissue kinetics of colorectal carcinoma cells in hepatic metastases are biologically quite different from those of primary tumors, probably because of co-operative effects of both internal (AI, survivin, bcl-2) and external (IMVD) regulation factors.

Adenocarcinoma↗