Biomedical subjects

J A Roth

Publications and source records attributed to J A Roth.

At least 55 records · Page 3Linked to original sources

PubMed2001

P53 gene replacement for cancer--interactions with DNA damaging agents.

Clinical trials of p53 gene replacement have provided information that will be useful in the design of future gene therapy strategies. Direct intratumor injection has low toxicity and thus can be readily combined with existing treatments. Post-injection gene expression can be documented and occurs in the presence of an anti-adenovirus immune response. Importantly, this treatment can cause tumor regression or prolonged stabilization. Future research directions will include development of more efficient vectors, use of novel genes, and combined modality approaches. Unresectable tumors are a prominent problem in oncology, with proven therapies such as radiotherapy and chemotherapy controlling less than 20% of lung cancers. Based on the preclinical and clinical studies discussed, it now seems that these conventional therapies may provide renewed potential when used in conjunction with transfer of a functional p53 gene.

Adenoviridae↗

PubMed2001

Adenovirus-mediated wild-type p53 gene expression radiosensitizes non-small cell lung cancer cells but not normal lung fibroblasts.

PURPOSE: We compared the ability of adenoviral-mediated wild-type p53 RPR/INGN201(Ad5/CMV/p53) to radiosensitize non-small cell lung carcinoma (NSCLC) and normal lung fibroblast cells. MATERIALS AND METHODS: NSCLC cell lines (A549 and H322) and human lung fibroblast cells (MRC-9 and CCD-16) were used in this study. Radiosensitivity was determined by clonogenic assay and tumor growth delay. Expression of p53, Bax, and p21WAF1 protein were evaluated by immunoblot. A FITC conjugate of annexin V was used for flow cytometric detection of apoptosis. RESULTS: Clonogenic and apoptotic assays indicated that Ad5/CMV/p53 enhanced the radiosensitivity of both NSCLC cell lines. On the other hand, the two normal human fibroblast cell lines appeared to be resistant to the cytotoxic effects of Ad5/CMV/p53 and were not radiosensitized compared to the NSCLC cells. According to immunoblot analysis, Bax expression was increased in the NSCLC cells treated with the combination therapy; Bax expression, however, was unchanged in normal cells. In in vivo studies, tumor growth suppression was enhanced by this combination strategy in xenograft tumors growing in nude mice compared to Ad5/CMV/p53 or radiation therapy when used alone. CONCLUSIONS: Our data indicate that therapy using Ad5/CMV/p53 and irradiation in combination is more effective than either treatment when used alone on NSCLC cells, is not limited to cells with defective endogenous p53, and does not enhance the radiosensitivity of normal cells.

Adenoviridae↗

PubMed2001

Pilot study of concurrent 5-fluorouracil/paclitaxel plus radiotherapy in patients with carcinoma of the esophagus and gastroesophageal junction.

Preoperative concurrent chemotherapy and radiotherapy can be highly effective but are often associated with significant rates of morbidity and even mortality. We studied the toxicity of continuous infusion of 5-fluorouracil (5-FU) and weekly paclitaxel combined with radiotherapy. Patients had histologic proof of local-regional carcinoma of the esophagus or gastroesophageal (GE) junction, a Karnofsky performance status of 70 or greater, and normal liver, renal, and bone marrow functions. Chemotherapy consisted of continuous infusion of 5-FU (300 mg/m2/d) for 5 days a week for 5 weeks, plus paclitaxel (45 mg/m2) given during 3 hours every week for 5 weeks. Based on the tumor location and its resectability, the total dose of concurrent radiation varied between 45 Gy and 50.4 Gy. Nine men and one woman, with a median age of 61 years, were evaluated. One had GE junction cancer, six had distal esophageal cancer, and three had midesophageal cancer. Weight loss, nausea, vomiting, and dysphagia of grades I and II were noted. The hematologic toxicity was mild. No patients required transfusion. There was no leukopenia or thrombocytopenia. None of the patients was hospitalized during chemoradiation; all patients completed treatment as outpatients. Five patients had subsequent surgical resections: one had a pathologically complete response, and two had a partial response (>90% necrosis). Continuous infusion of 5-FU plus paclitaxel given concurrently with radiotherapy was well tolerated. We plan to study this regimen further in upper gastrointestinal cancers.

Adenocarcinoma↗

PubMed2001

Prenatal hydronephrosis.

Prenatal hydronephrosis is diagnosed with an incidence of 1:100 to 1:500 maternal-fetal ultrasonographic studies. Although ultrasonography accurately describes dilation of the renal collecting system, it cannot define mechanical obstruction, a functional characteristic. The purpose of prenatal screening has changed from simple detection of hydronephrosis to selection for specific diagnosis-based management. The natural history of prenatal hydronephrosis is difficult to determine, and therefore physicians who take care of the fetus and infant are challenged with the following question: when does dilation of the renal collecting system indicate that serious pathology (ongoing renal deterioration) exists? The diagnosis of prenatal hydronephrosis, indications for and timing of prenatal intervention, and recommendations for treating children with a history of prenatal hydronephrosis are included in this review.

Female↗

PubMed2001

Enhancement of neutrophil function by ultrafiltered bovine whey.

The objective of this work was to evaluate a proprietary ultrafiltered bovine whey product for its in vitro influence on the function of neutrophils from normal and dexamethasone-treated cattle, and for its in vivo influence on neutrophil function in periparturient dairy cows. The ultrafiltered bovine whey was produced by hyperimmunizing cows to various bacterial pathogens by intramammary injection, collecting and pooling the colostrum and milk for the first 3 wk after parturition, then separating and processing the whey through a filter with a nominal molecular mass cut off of 10,000 Da. In vitro treatment of neutrophils from normal calves with ultrafiltered bovine whey significantly increased neutrophil random migration, cytochrome C reduction, iodination activity, antibody-dependent cell-mediated cytotoxicity, and antibody-independent cell-mediated cytotoxicity. Cytochrome C reduction was the only neutrophil function parameter significantly enhanced by the in vitro treatment of neutrophils from dexamethasone-treated cattle with ultrafiltered bovine whey. In vivo treatment of periparturient cows with ultrafiltered bovine whey did not alter the total or differential leukocyte counts in the animals but did significantly increase the total erythrocyte counts. In vivo treatment with ultrafiltered bovine whey also significantly increased neutrophil iodination activity in the periparturient cows. Neutrophil iodination activity (a measure of the myeloperoxidase/hydrogen peroxide/halide antibacterial system) is a very potent bactericidal mechanism of neutrophils and has previously been shown to be suppressed in periparturient cows.

Animals↗

PubMed2001

Adenovirus-mediated antisense urokinase-type plasminogen activator receptor gene transfer reduces tumor cell invasion and metastasis in non-small cell lung cancer cell lines.

The urokinase-type plasminogen activator (uPA) and its receptor (uPAR) play an important role in the proteolytic cascade involved in the metastasis of lung and other cancers. We report that the reduction in uPAR levels produced by an antisense strategy using an adenovirus construct (Ad-uPAR) in H1299 cells, an invasive human lung cancer cell line that produces high levels of uPAR, resulted in a decrease of uPAR levels to 80-90% of those seen in cells infected with mock or adenovirus (Ad)-cytomegalovirus vector controls. In addition, increasing the multiplicity of infection from 25 to 200 caused a corresponding decrease in the level of uPAR protein within 5 days of treatment, as shown by Western blot analysis. Furthermore, the in vitro translation of total RNA levels of Ad-uPAR-infected H1299 cells in a rabbit reticulocyte lysate system caused a 50-70% decrease in uPAR immunoprecipitate in Ad-uPAR-infected cells relative to the levels in cells of mock and vector controls. The Matrigel invasion assay showed the invasion of H1299 cells and A549 cells infected with Ad-uPAR to be decreased by 70% relative to mock- and vector-infected controls. Infection of tumor cells with Ad-uPAR before implantation significantly reduced the incidence of lung metastasis by 85% as compared with the control virus-infected cells injected into nude mice through the tail vein. Our collective results show that the uPAR system is a potential target of treatment for lung cancers.

Adenoviridae↗

PubMed2001

Deficiency of caspase-3 in MCF7 cells blocks Bax-mediated nuclear fragmentation but not cell death.

Caspase-3 plays a critical role in a proteolytic cascade within the apoptosis signal pathway; this enzyme is commonly activated by numerous death signals and cleaves a variety of important cellular proteins. Using caspase-3-deficient MCF7 cells and clones stably transfected with the caspase-3 gene (MCF7/Casp3), we evaluated the role of caspase-3 in Bax-induced apoptosis. Bax overexpression induced cell death in both parental MCF7 cells and MCF7/Casp3 cells. The introduction of the caspase-3 gene did not change the rate of cell death. Caspase-3-deficient parental MCF7 cells, however, failed to undergo morphological nuclear and DNA fragmentation, whereas MCF7/casp3 cells displayed intact nuclear dismantling and DNA fragmentation. Caspase-3 deficiency, however, did not affect Bax-induced levels of poly(ADP-ribose) polymerase cleavage, caspase-6 activation, and lamin B cleavage. Together, these results suggest that a deficit in caspase-3 is not sufficient to block Bax-induced cell death.

Apoptosis↗

PubMed2001

Melanoma differentiation associated gene-7 (mda-7): a novel anti-tumor gene for cancer gene therapy.

BACKGROUND: The mda-7 gene (melanoma differentiation associated gene-7) is a novel tumor suppressor gene. The anti-proliferative activity of MDA-7 has been previously reported. In this report, we analyze the anti-tumor efficacy of Ad-mda7 in a broad spectrum of cancer lines. MATERIALS AND METHODS: Ad-mda7-transduced cancer or normal cell lines were assayed for cell proliferation (tritiated thymidine incorporation assay, Alamar blue assay, and trypan-blue exclusion assay), apoptosis (TUNEL, and Annexin V staining visualized by fluorescent microscopy or FACs analysis), and cell cycle regulation (Propidium Iodide staining and FACs analysis). RESULTS: Ad-mda7 treatment of tumor cells resulted in growth inhibition and apoptosis in a temporal and dose-dependent manner. The anti-tumor effects were independent of the genomic status of p53, RB, p16, ras, bax, and caspase 3 in these cells. In addition, normal cell lines did not show inhibition of proliferation or apoptotic response to Ad-mda7. Moreover, Ad-mda7-transduced cancer cells secreted a soluble form of MDA-7 protein. Thus, Ad-mda7 may represent a novel gene-therapeutic agent for the treatment of a variety of cancers. CONCLUSIONS: The potent and selective killing activity of Ad-mda7 in cancer cells but not in normal cells makes this vector a potential candidate for cancer gene therapy.

Adenoviridae↗

PubMed2001

Synergistic inhibition of human lung cancer cell growth by adenovirus-mediated wild-type p53 gene transfer in combination with docetaxel and radiation therapeutics in vitro and in vivo.

Chemotherapy given sequentially or concurrently with external beam radiation therapy has emerged as a standard for the treatment of locally advanced lung cancer. Gene therapy by adenovirus-mediated wild-type p53 gene transfer has been shown to inhibit lung cancer growth in vitro, in animal models, and in human clinical trials. However, no information is available on the combined effects of p53 gene transfer, chemotherapy, and radiation therapy on lung cancer growth in vitro and in vivo. Therefore, we developed two-dimensional and three-dimensional isobologram modeling and statistical methods to evaluate the synergistic, additive, or antagonistic efficacy among these therapeutic agents in human non-small cell lung cancer cell lines A549, H460, H322, and H1299, at the ID50 and ID80 levels. The combination of these three therapeutic agents exhibited synergistic inhibitory effects on tumor cell growth in all four cell lines at both the ID50 and the ID80 levels in vitro. In mouse models with H1299 and A549 xenografts, combined treatment synergistically inhibited tumor growth in the absence of any apparent increase in toxicity, when compared with other treatment and control groups. Together, our findings suggest that a combination of gene therapy, chemotherapy, and radiation therapy may be an effective strategy for human cancer treatment.

Adenoviridae↗

PubMed2000

Manganese induces neurite outgrowth in PC12 cells via upregulation of alpha(v) integrins.

Previous studies have demonstrated that the divalent cation manganese (Mn) causes PC12 cells to form neurites in the absence of NGF. Since divalent cations modulate the binding affinity and specificity of integrins, and integrin function affects neurite outgrowth, we tested the hypothesis that Mn induces neurite outgrowth through an integrin-dependent signaling pathway. Our studies support this hypothesis. Function-blocking antisera specific for beta(1) integrins block the neurite-promoting activity of Mn by 90-95%. Bioassays and biochemical studies with antisera specific for the alpha(v), alpha(5), or alpha(8) integrin subunit suggest that the alpha(v)beta(1) heterodimer is one of the principal beta(1) integrins mediating the response of PC12 cells to Mn. This is corroborated by studies in which Mn failed to induce neurite outgrowth in a clone of PC12 cells that does not express alpha(v) at levels detectable by immunoprecipitation or immunocytochemistry. SDS-PAGE analysis of biotinylated surface proteins immunoprecipitated from Mn-responsive PC12 cells, as well as confocal laser microscopy of PC12 immunostained for surface alpha(v) indicate that Mn increases the surface expression of alpha(v) integrins. This increase appears to be due in part to synthesis of alpha(v) since specific inhibitors of RNA and protein synthesis block the neurite-promoting activity of Mn. These data indicate that Mn induces neurite outgrowth in PC12 cells by upregulating alpha(v) integrins, suggesting that Mn potentially represents an additional mechanism for regulating the rate and direction of neurite outgrowth during development and regeneration.

Animals↗

PubMed2000

Contribution of CD95 ligand-induced neutrophil infiltration to the bystander effect in p53 gene therapy for human cancer.

Clinical trials of adenoviral p53 gene therapy provide the evidence that the bystander effect induced by the wild-type p53 gene transfer on adjacent tumor cells contributes to tumor progression; its mechanism, however, remains uncharacterized. We report in this work that injection of adenovirus expressing the human wild-type p53 gene (Ad5CMVp53) into established human colorectal tumors in nu/nu mice resulted in CD95 ligand (CD95L) overexpression, followed by a massive neutrophil infiltration. Culture supernatants of human colorectal cancer cells infected with Ad5CMVp53 exhibited a potent chemotactic activity against murine polymorphonuclear neutrophils, which could be abolished by the anti-CD95L mAb (NOK-1). In vivo cell depletion experiments indicated that neutrophils were in part responsible for the antitumor effect of the Ad5CMVp53 infection. Our data directly suggest that overexpression of CD95L by the wild-type p53 gene transfer induces neutrophil infiltration into human colorectal tumors, which may play a critical role in the bystander effect of p53 gene therapy.

Adenoviruses, Human↗

PubMed2000

Adenovirus-mediated p16INK4a gene expression radiosensitizes non-small cell lung cancer cells in a p53-dependent manner.

We examined the influence of adenovirus-mediated wild-type p16INK4a (Ad/p16) expression on the radiation sensitivity of NSCLC cell lines, all of which lacked constitutive p16INK4a but each of which varied in p53 status: A549 (-p16INK4a/ +pRb/wt-p53), H322 (-p16INK4a/ +pRb/mt-p53), and H1299 (-p16INK4a/ +pRb/deleted-p53). The in vitro clonogenic survival results indicate that Ad/p16 enhanced the radiosensitivity of A549 but not H322 or H1299. Further analysis indicated that the apoptosis induced by combination therapy using Ad/p16 plus irradiation was dependent on the endogenous p53 status of the cancer cells. We performed Western blotting to analyse the p53 protein expression of A549 cells treated with either Ad/p16 or Ad/Luc. Endogenous p53 protein levels were higher in A549 cells transfected with Ad/p16 than in those transfected with Ad/Luc. Furthermore, when wt-p53 protein expression was restored in H1299 using Ad/ p53, Ad/p16 stabilized p53 protein expression and radiosensitized the cells. These results suggest that Ad/ p16-induced stabilization of p53 protein may play an important role in Ad/p16 mediated radiosensitization by enhancing or restoring apoptosis properties. Thus, Ad/ p16 plus radiation in combination may be a useful gene therapy strategy for tumors that have wt-p53 but nonfunctional p16INK4a.

Adenoviruses, Human↗

PubMed2000

Oral administration of an estrogen metabolite-induced potentiation of radiation antitumor effects in presence of wild-type p53 in non-small-cell lung cancer.

PURPOSE: The purpose of this study was to investigate the efficacy of 2-methoxyestradiol as an antitumor and radiosensitizing agent for the treatment of human malignancy. METHODS AND MATERIALS: Two cancer cell lines with wild-type p53 status were exposed first to irradiation and then to an oral formulation of the nontoxic metabolite 2-methoxyestradiol (2ME) to stabilize p53 levels. RESULTS: Cell growth was inhibited via G1 growth and apoptosis. Subsequent in vitro growth and Tunel assays indicated that this combination was superior to radiation alone at inducing p53 protein accumulation, stabilizing p53 protein levels, and substantially reducing long-term tumor cell growth (approximately 80%) and colony formation (approximately 95%) in vitro, and inducing apoptosis. However, harboring mutated p53, H322 cell line, was relatively insensitive to such a treatment regimen. Western blot analysis revealed that growth inhibition was associated with increased levels of p53 and p21 protein accumulation. Experiments with subcutaneous tumor in a nu/nu mouse showed the combination treatment to be superior to radiation alone at reducing tumor growth ( approximately 50% reduction as compared to radiation alone) in vivo. CONCLUSION: Thus, our studies confirmed a unique strategy whereby oral administration of a nontoxic estrogen metabolite, 2ME, significantly enhanced the radiation effect on a subcutaneous tumor without any toxicity and suggesting that this strategy may be clinically useful as an adjuvant therapy.

2-Methoxyestradiol↗

PubMed2000

Differential localization of divalent metal transporter 1 with and without iron response element in rat PC12 and sympathetic neuronal cells.

Two isoforms of divalent metal transporter 1 (DMT1) (Nramp2 and DCT1) are encoded by two mRNA species, one of which contains an iron response element (IRE) motif in the 3'-noncoding region. The subcellular distribution of the two isoforms of DMT1 is distinct, and the -IRE species accumulates in the nucleus of neuronal or neuronal-like cells. Reverse transcription-PCR and Western blot analysis of PC12 cells reveals that these cells express both forms of DMT1. Immunofluorescence and immunoblotting studies, using immunospecific antibodies to the -IRE form of DMT1, demonstrate that this form of the transporter, in PC12 cells, is predominantly localized in the nucleus, cell membrane, and neurites with only weak staining of the cell body. Studies using antibodies to the +IRE form indicate that this species of DMT1 is distributed within vesicles in the cell body and neurite projections, with minimal nuclear staining. Similar staining patterns are observed for the two forms of DMT1 in cultures of sympathetic ganglion neurons isolated from perinatal rat pups. To determine whether nuclear localization of the -IRE form of DMT1 is constrained to neuronal or neuronal-like cells, immunocytochemical studies were performed with human embryonic kidney 293T (HEK293T), HEP2G hepatoma and medulloblastoma, and rat Schwann cells. The -IRE-specific antibodies stained nuclei from medulloblastoma, whereas little nuclear staining was observed with HEK293T, hepatoma, or Schwann cells. The unexpected finding that the -IRE species of DMT1 selectively accumulates in the nucleus of neuronal and neuronal-like cells leads us to postulate that the two proteins may have different functions in vivo.

Animals↗

PubMed2000

Tumor-specific transgene expression from the human telomerase reverse transcriptase promoter enables targeting of the therapeutic effects of the Bax gene to cancers.

Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of telomerase, which is highly active in immortalized cells and >85% of human cancers but is quiescent in most normal somatic cells. To test the feasibility of using the hTERT promoter to induce tumor-specific transgene expression in cancer gene therapy, we constructed an adenoviral vector expressing a LacZ reporter gene driven by the hTERT core promoter and evaluated its activity in vitro and in vivo. The hTERT promoter could drive high-level expression of LacZ in tumor cells but not in normal cells and normal mouse tissues. Using a binary adenoviral system that can induce Bax gene expression, we showed that induction of the Bax gene expression via the hTERT promoter elicited tumor-specific apoptosis in vitro, suppressed tumor growth in nude mice, and prevented the toxicity of the Bax gene in vitro and in vivo. Thus, the hTERT promoter is apparently a strong and tumor-selective promoter with potential application in targeted cancer gene therapy.

Adenoviridae↗

PubMed2000

Insulin-like growth factor binding protein-6 activates programmed cell death in non-small cell lung cancer cells.

Insulin-like growth factor binding proteins (IGFBPs) are secreted into the extra-cellular matrix and inhibit cell growth through IGF-dependent and -independent mechanisms. In this study, we investigated the role of IGFBP-6, a relatively unexplored member of the IGFBP family, in the proliferation of non-small cell lung cancer (NSCLC) cells. Infection of NSCLC cell lines in vitro with an adenovirus expressing human IGFBP-6 under the control of a CMV promoter (Ad5CMV-BP6) reduced NSCLC cell number through activation of programmed cell death, as shown by cell staining with Hoechst 33342 or DNA end-labeling with bromodeoxyuridine triphosphate. The growth regulatory effect of IGFBP-6 was investigated in vivo by intratumoral injection of Ad5CMV-BP6 in NSCLC xenografts established in nu/nu mice. A single injection of Ad5CMV-BP6 reduced the size of NSCLC xenografts by 45%. These findings indicate that IGFBP-6 is a potent inducer of programmed cell death in cancer cells and support investigations into IGFBP-6 as a potential target in cancer therapeutics.

Adenoviridae↗

PubMed2000

Outcome predictors for 143 patients with superior sulcus tumors treated by multidisciplinary approach at the University of Texas M. D. Anderson Cancer Center.

PURPOSE: Superior sulcus tumors (SST) of the lung are uncommon and constitute approximately 3% of non-small cell lung cancer (NSCLC). These tumors cause specific symptoms and signs, and are associated with patterns of failure that differ from those seen for NSCLC tumors in other nonapical locations. Prognostic factors and most effective treatments are controversial. We conducted a retrospective study at The University of Texas M. D. Anderson Cancer Center to identify outcome predictors for patients with SST treated by a multidisciplinary approach. METHODS AND MATERIALS: This retrospective review of 143 patients without distant metastasis at presentation is a continuation of a previous M. D. Anderson study now updated to 1994. In this study, we examine the 5-year survival rate by pretreatment tumor and patient characteristics and by the treatments received. Strict criteria were used to define SST. Actuarial life-table analyses and Cox proportional hazard models were used to compare survival rates. RESULTS: Overall predictors of 5-year survival were weight loss (p < 0.01), supraclavicular fossa (p = 0. 03), or vertebral body (p = 0.05) involvement, stage of the disease (p < 0.01), and surgical treatment (p < 0.01). Five-year survival for patients with Stage IIB disease was 47% compared to 14% for Stage IIIA, and 16% for Stage IIIB. For patients with Stage IIB disease, surgical treatment (p < 0.01) and weight loss (p = 0.01) were significant independent predictors of 5-year survival. Among patients with Stage IIIA disease, the only predictor of survival was Karnofsky performance score (KPS) (p = 0.02). For patients with Stage IIIB disease, the only independent predictor of survival was a right superior sulcus location, which was associated with a worse 5-year survival rate than that for patients with tumors in the left superior sulcus (p = 0.02). More patients with adenocarcinoma than with squamous cell tumors experienced cerebral metastases within 5 years (p < 0.01). Patients without gross residual disease after surgical resection who received postoperative radiation therapy with total doses of 55 to 64 Gy had a 5-year survival rate of 82% as compared with the 5-year survival rate of 56% in patients who received 50 to 54 Gy. Twenty-three patients survived for longer than 3 years. Of these, 4 patients (17%) received radiation therapy alone or in combination with chemotherapy without surgical resection. The other 19 patients (83%) had resection combined with radiation therapy and/or chemotherapy. CONCLUSIONS: The findings from this study confirm the importance of the new staging system, separating T3 N0 M0 (Stage IIB) from Stage IIIA, since there was a significant difference in the 5-year survival (p < 0.01). Interestingly, there was no significant 5-year survival difference between Stage IIIA (N2) and Stage IIIB (T4 or N3). This study also suggests that surgery is an important component of the multidisciplinary approach to patients with SST if their nodes were negative. Disease that is minimally invading surrounding normal structures can be resected followed by radiation therapy in doses of 55 to 64 Gy. Further investigation of treatment strategies combining high-dose radiation therapy (>/=66 Gy) with chemotherapy is indicated for patients with unresectable and/or node-positive (N2) SST.

Adult↗

PubMed2000

Manganese-induced rat pheochromocytoma (PC12) cell death is independent of caspase activation.

Manganese (Mn) is an essential mineral that at high concentrations can produce an irreversible syndrome resembling Parkinson's disease. To examine the mechanism by which Mn elicits its toxic response, we have selected the rat pheochromocytoma cells (PC12) as our model system because it possesses much of the biochemical machinery associated with dopaminergic neurons. Mn-induced PC12 cell death is both time and concentration dependent with approximately 50% cell survival at 48 hr in the presence of 0.3 mM Mn. To determine whether oxidative stress contributed to cytotoxicity induced by Mn, lipid peroxidation was assessed in Mn-treated in PC12 cells. The highly sensitive HPLC assay that measures the lipid peroxide product, 9-HODE, was used and results of these experiments demonstrate there was no increase in the lipid peroxidation in cells exposed to 0.3 mM Mn for 24 hr. Mn was found to stimulate the activation of the apoptotic marker proteins, p38 and caspase-3 within the first 24 hr of treatment. The selective inhibitor of caspase-3, DEVD-CHO, and the nonselective caspase inhibitor, Z-VAD-FMK, however, fail to prevent Mn-induced PC12 cell death. Studies were performed to determine the role of mitochondria in initiating or supporting Mn cytotoxicity, because Mn has been reported to cause changes in membrane permeability. Mn caused a decrease in ATP levels in PC12 cells in both a time and concentration dependent manner. We hypothesize that both apoptosis and necrosis contribute to PC12 cell death although the necrotic events prevail even when the apoptotic signaling is inhibited.

Animals↗