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Biomedical subjects

E J Bernhard

Publications and source records attributed to E J Bernhard.

At least 19 recordsLinked to original sources
PubMed2001

Tissue specific expression of p53 target genes suggests a key role for KILLER/DR5 in p53-dependent apoptosis in vivo.

The p53 tumor suppressor plays a key role in the cell's response to genotoxic stress and loss of this 'guardian of the genome' is an important step in carcinogenesis. The ability of p53 to induce apoptosis through transactivation of its target genes is critical for its function as tumor suppressor. We have found that overexpression of p53 in human cancer cell lines resulted in apoptosis as measured by PARP cleavage. Furthermore we observed cleavage of both caspase 9 and caspase 8 after overexpression of p53 and found that p53-dependent apoptosis was inhibited by either cellular (c-Flip-s, Bcl-X(L)) or pharmacological inhibitors of caspase 8 or caspase 9 respectively. These results indicate that p53 is mediating apoptosis through both the mitochondrial and death receptor pathways. To elucidate the relevant p53 target genes and examine the caspase pathways utilized in vivo, we treated p53+/+ and age matched p53-/- mice with 5 Gy ionizing radiation or 0.5 mg/animal dexamethasone and harvested tissues at 0, 6 and 24 h. We examined the mRNA expression of p21, bax, KILLER/DR5, FAS/APO1 and EI24/PIG8 using TaqMan real time quantitative RT-PCR in the spleen, thymus and small intestine. Although the basal mRNA levels of these genes did not depend on the presence of p53, we observed a p53-dependent induction of all these targets in response to gamma-irradiation and a p53-independent regulation for p21 and KILLER/DR5 in response to dexamethasone. Furthermore, we have demonstrated that the relative induction of these p53 target genes is tissue specific. Despite observing otherwise similar levels of death in these tissues, our findings suggest that in some cases apoptosis mediated through p53 occurs by redundant pathways or by a 'group effect' while in other tissues one or few targets may play a key role in p53-dependent apoptosis. Surprisingly, KILLER/DR5 is the dominantly induced transcript in both the spleen and small intestine suggesting a potentially important role for this p53 target gene in vivo.

Animals↗
PubMed2001

RhoB is required to mediate apoptosis in neoplastically transformed cells after DNA damage.

The effect of neoplastic transformation on the response to genotoxic stress is of significant clinical interest. In this study, we offer genetic evidence that the apoptotic response of neoplastically transformed cells to DNA damage requires RhoB, a member of the Rho family of actin cytoskeletal regulators. Targeted deletion of the rhoB gene did not affect cell cycle arrest in either normal or transformed cells after exposure to doxorubicin or gamma irradiation, but rendered transformed cells resistant to apoptosis. This effect was specific insofar as rhoB deletion did not affect apoptotic susceptibility to agents that do not damage DNA. However, rhoB deletion also affected apoptotic susceptibility to Taxol, an agent that disrupts microtubule dynamics. We have demonstrated that RhoB alteration mediates the proapoptotic and antineoplastic effects of farnesyltransferase inhibitors, and we show here that RhoB alteration is also crucial for farnesyltransferase inhibitors to sensitize neoplastic cells to DNA damage-induced cell death. We found RhoB to be an important determinant of long-term survival in vitro and tumor response in vivo after gamma irradiation. Our findings identify a pivotal role for RhoB in the apoptotic response of neoplastic cells to DNA damage at a novel regulatory point that may involve the actin cytoskeleton.

Alkyl and Aryl Transferases↗
PubMed2001

The Ras radiation resistance pathway.

The critical pathways determining the resistance of tumor cells to ionizing radiation are poorly defined. Because the ras oncogene, a gene activated in many human cancers treated with radiotherapy, can induce increased radioresistance, we have asked which Ras effector pathways are significant in conferring a survival advantage to tumor cells. The phosphoinositide-3-kinase (PI3K) inhibitor LY294002 radiosensitized cells bearing mutant ras oncogenes, but the survival of cells with wild-type ras was not affected. Inhibition of the PI3K downstream target p70S6K by rapamycin, the Raf-MEK-MAPK pathway with PD98059, or the Ras-MEK kinase-p38 pathway with SB203580 had no effect on radiation survival in cells with oncogenic ras. Expression of active PI3K in cells with wild-type ras resulted in increased radiation resistance that could be inhibited by LY294002. These experiments have indicated the importance of PI3K in mediating enhanced radioresistance and have implicated PI3K as a potential target for specific radiosensitization of tumors.

Animals↗
PubMed2001

The farnesyltransferase inhibitor L744,832 reduces hypoxia in tumors expressing activated H-ras.

Many tumors contain extensive regions of hypoxia. Because hypoxic cells are markedly more resistant to killing by radiation, repeated attempts have been made to improve the oxygenation of tumors to enhance radiotherapy. We have studied the oxygenation of tumor xenografts in nude mice after treatment with the farnesyltransferase inhibitor L744,832. Hypoxia was assessed by measuring the binding of the hypoxic cell marker pentafluorinated 2-nitroimidazole. We show that xenografts from two tumor cell lines with mutations in H-ras had markedly improved oxygenation after farnesyltransferase treatment. In contrast, xenografts from two tumors without ras mutations had equivalent hypoxia regardless of treatment. The effect on tumor oxygenation could be detected at 3 days and remained after 7 days of treatment. These results indicate that treatment with farnesyltransferase inhibitors can alter the oxygenation of certain tumors and suggest that such treatment might be useful in the radiosensitization of these tumors.

Alkyl and Aryl Transferases↗
PubMed2001

Apoptosis: an early event in metastatic inefficiency.

Whereas large numbers of cells from a primary tumor may gain access to the circulation, few of them will give rise to metastases. The mechanism of elimination of these tumor cells, often termed "metastatic inefficiency," is poorly understood. In this study, we show that apoptosis in the lungs within 1-2 days of introduction of the cells is an important component of metastatic inefficiency. First, we show that death of transformed, metastatic rat embryo cells occurred via apoptosis in the lungs 24-48 h after injection into the circulation. Second, we show that Bcl-2 overexpression in these cells inhibited apoptosis in culture and also conferred resistance to apoptosis in vivo in the lungs 24-48 h after injection. This inhibition of apoptosis led to significantly more macroscopic metastases. Third, comparison between the extent of apoptosis by a poorly metastatic cell line to that by a highly metastatic cell line 24 h after injection in the lungs revealed more apoptosis by the poorly metastatic cell line. These results indicate that apoptosis, which occurs at 24-48 h after hematogenous dissemination in the lungs is an important determinant of metastatic inefficiency. Although prior work has shown an association between apoptosis in culture and metastasis in vivo, this work shows that apoptosis in vivo corresponds to decreased metastasis in vivo.

Animals↗
PubMed2000

Direct evidence for the contribution of activated N-ras and K-ras oncogenes to increased intrinsic radiation resistance in human tumor cell lines.

Transformation with ras oncogenes results in increased radiation sur vival in many but not all cells. In addition, prenyltransferase inhibitors which inhibit ras proteins by blocking posttranslational modification radiosensitize cells with oncogenic ras. These findings suggest that oncogenic ras contributes to intrinsic radiation resistance. However, because introduction of ras oncogenes does not increase radiation survival in all cells and because prenyltransferase inhibitors target molecules other than ras, these studies left the conclusion that ras increases the intrinsic radi ation resistance of tumor cells in doubt. Here we show that genetic inactivation of K- or N-ras oncogenes in human tumor cells (DLD-1 and HT1080, respectively) leads to increased radiosensitivity. Reintroduction of the activated N-ras gene into the HT1080 line, having lost its mutant allele, resulted in increased radiation resistance. This study lends further support to the hypothesis that expression of activated ras can contribute to intrinsic radiation resistance in human tumor cells and extends this finding to the K- and N- members of the ras family. These findings support the development of strategies that target ras for inactivation in the treatment of cancer.

Adenocarcinoma↗
PubMed2000

Re-evaluating gadolinium(III) texaphyrin as a radiosensitizing agent.

Gadolinium(III) texaphyrin (Gd-tex) was recently proposed as a radiosensitizing agent that combines preferential tumor uptake with detection of drug localization by magnetic resonance imaging (S. W. Young et al., Proc. Natl. Acad. Sci. USA, 93: 6610-6615, 1996). In view of the initial report on this compound, four radiobiology laboratories undertook independent efforts to further study radiosensitization by Gd-tex. In addition to repeating the previously reported studies on Gd-tex in HT-29 cells, we tested five other human tumor cell lines (U-87 MG, U251-NCI, SW480, A549, and MCF-7). These studies included a Gd-tex treatment period of 24 h before irradiation (as in the original publication), with concentrations of Gd-tex ranging from 20-500 microM. In neither the HT-29 cells nor any of the other five human cell lines did we see radiation sensitization by Gd-tex. Two cell lines (MCF-7 and U-87 MG) were further tested for radiosensitization by Gd-tex under hypoxic conditions. No radiosensitization was observed in either case. Finally, the radiation response of two tumor lines were assessed in vivo. Neither HT-29 xenografts in severe combined immunodeficient (SCID) mice nor RIF-1 tumors growing in C3H mice demonstrated radiosensitization after Gd-tex treatment before single or fractionated doses of radiation. Our results raise questions about the efficacy of Gd-tex as a radiosensitizing agent.

Animals↗
PubMed2000

DNA as an important target in radiation-induced apoptosis of MYC and MYC plus RAS transfected rat embryo fibroblasts.

PURPOSE: This study uses a radiation chemistry approach to determine if DNA is an important target for radiation-induced apoptosis of myc (MR4) and myc plus ras (3.7) transfected rat embryo fibroblast cell lines. MATERIALS AND METHODS: The radiation protection efficiency of four thiols was compared with net molecular charge ranging from -1 to +2: mercaptopropionic acid (Z= -1), mercaptoethanol (Z=0), cysteamine (Z= +1), N(2-mercaptoethyl)-1,3-diaminopropane (Z= +2). Protection factors were determined for these thiols against radiation-induced apoptosis (Apoalert assay), mitotic cell death (clonogenic assay) and double-strand break (dsb) induction (pulse field gel electrophoresis) in MR4 and 3.7 cells. Theoretical protection factors for these thiols against dsb induction were also calculated from second-order chemical repair constants for single-strand breaks (ssb) and the concentration of added thiols in MR4 and 3.7 cell lines. RESULTS: The charge-dependent increases observed for measured protection factors against radiation-induced apoptosis did not differ significantly between the two cell lines, nor did they differ significantly from the corresponding increases observed for radiation-induced mitotic cell killing and for induction of dsb. The calculated protection factor for dsb also showed a thiol charge-dependent increase similar to the measured protection factors for all of the other parameters studied. CONCLUSIONS: These results are consistent with the hypothesis that DNA is an important target for radiation-induced apoptosis.

Animals↗
PubMed2000

RAS-Mediated radiation resistance is not linked to MAP kinase activation in two bladder carcinoma cell lines.

The expression of activated RAS oncogenes has been shown to increase radioresistance in a number of cell lines. The pathways by which RAS leads to radioresistance, however, are unknown. RAS activates several signal transduction pathways, with the RAF-MAP2K-MAP kinase pathway perhaps the best studied. MAP kinase has also been shown to be activated by radiation through this pathway. Given the important role of MAP kinase in multiple signaling events, we asked if radioresistance induced by RAS was mediated through the activation of MAPK. Cells of two human bladder carcinoma cell lines were used, one with a mutated oncogenic HRAS (T24) and other with a wild-type HRAS (RT4). The surviving fraction after exposure to 2 Gy of radiation (SF2) for the T24 cell lines was found to be 0.62, whereas that for RT4 cells was 0.40. Treatment with the farnesyl transferase inhibitor (FTI) L744,832, which inhibits RAS processing and activity, decreased the SF2 of T24 cells to 0.29, whereas the SF2 of RT4 cells remained unchanged after FTI treatment, thus demonstrating the importance of RAS activation to the radiosensitivity of cells with mutated RAS. MAP kinase activation was found to be constitutive and dependent on RAS in T24 cells, while it was inducible by radiation and was independent of RAS in RT4 cells. Treatment of both cell lines with the MAP2K inhibitor PD98059 inhibited MAPK activation; however, inhibiting MAPK activation had no effect on radiation survival of T24 or RT4 cells. These data indicate that MAPK activation does not contribute to RAS-induced radioresistance in this system.

Cell Survival↗
PubMed2000

Farnesyltransferase inhibitors potentiate the antitumor effect of radiation on a human tumor xenograft expressing activated HRAS.

Successful radiosensitization requires that tumor cells become more radiosensitive without causing an equivalent reduction in the survival of cells of the surrounding normal tissues. Since tumor cell radiosensitivity can be influenced by RAS oncogene activation, we have hypothesized that inhibition of oncogenic RAS activity would lead to radiosensitization of tumors with activated RAS. We previously showed in tissue culture that prenyltransferase treatment of cells with activated RAS resulted in radiosensitization, whereas treatment of cells with wild-type RAS had no effect on radiation survival. Here we ask whether the findings obtained in vitro have applicability in vivo. We found that treatment of nude mice bearing T24 tumor cell xenografts with farnesyltransferase inhibitors resulted in a significant and synergistic reduction in tumor cell survival after irradiation. The regrowth of T24 tumors expressing activated RAS was also significantly prolonged by the addition of treatment with farnesyltransferase inhibitors compared to the regrowth after irradiation alone. In contrast, there was no effect on the radiosensitivity of HT-29 tumors expressing wild-type RAS. These results demonstrate that specific radiosensitization of tumors expressing activated RAS oncogenes can be obtained in vivo.

Alkyl and Aryl Transferases↗
PubMed1999

How does radiation kill cells?

Recent advances in the understanding of intracellular signaling after genotoxic injury have led to a better understanding of the pathways that influence radiation-induced cell death. Particular progress has been made in defining molecular controls of apoptosis and radiation-induced cell cycle arrest, as well as the possible role of telomerase activity in stabilizing DNA breaks.

Animals↗
PubMed1998

Radiosensitization and apoptosis.

The toxicity of radiation to living tissues was discovered soon after the discovery of radioactivity itself and this toxicity is the basis for cancer therapy with radiation. Although this mode of therapy is often effective, its success is far from assured. One major difficulty in the implementation of radiotherapy is that normal tissues are also sensitive to killing by radiation so that treatment is often limited by the tolerance of normal tissues for radiation. Thus methods that sensitize tumor cells while sparing normal tissues could potentially lead to greater success with radiation as a therapy. Oncogenes are frequently altered in tumors, but are not in normal tissue making them potential targets for altering radiosensitivity and apoptosis in tumors.

Animals↗
PubMed1998

The measurement of bioreductive capacity of tumor cells using methylene blue.

PURPOSE: Methylene blue (MB) can be used as an intracellular electron acceptor. The purpose of this study was to demonstrate the usefulness of MB for the determination of total bioreductive capacity of cell suspensions. METHODS AND MATERIALS: We measured oxygen consumption by Clark electrode and pentose cycle activity by release of 14CO2 from 1-14C-glucose. RESULTS: Methylene blue catalyzes the reaction of intracellular reductants NADPH, NADH, and reduced glutathione (GSH) with oxygen, causing the production of hydrogen peroxide. The reaction rate correlates with the negative charge of molecule (NADPH(-4) > NADH(-2) > GSH(-1)), suggesting that reaction with positively charged oxidized MB is the limiting step of the reaction. In a cellular system MB causes the electron flow from cellular endogenous substrates to oxygen. It is activated by the disruption of the NADP+/NADPH ratio due to several processes. These are direct oxidation of NADPH and GSH, the GSH peroxidase catalyzed reaction of GSH with H2O2, followed by NADPH oxidation by oxidized glutathione (GSSG). This results in increased cellular oxygen consumption and stimulation of the oxidative limb of pentose cycle (PC) in the presence of MB. The cellular effect of MB differs from other electron accepting drugs. Diamide and tert-butylhydroperoxide act as direct oxidants, while MB is an electron carrier to oxygen. Accordingly, MB shows the highest effect on PC activation and oxygen consumption. CONCLUSIONS: Our results indicate that MB may be used for the determination of the total bioreductive capacity of the cells, measured by oxygen consumption and PC activation.

Carbon Dioxide↗
PubMed1998

Caffeine and staurosporine enhance the cytotoxicity of cisplatin and camptothecin in human brain tumor cell lines.

Caffeine and staurosporine have been shown to attenuate G2 delay produced by DNA-damaging agents and to augment the cytotoxicity of these agents in a number of cell lines in vitro. Studies in rodent brain tumor cell lines suggest that modulation of the G2/M transition may not contribute to the enhanced cytotoxicity produced by caffeine in brain tumor cells. To evaluate the impact of agents that decrease G2 delay on the cytotoxicity of chemotherapy in human brain tumor cells, we examined the ability of caffeine and staurosporine to modulate the G2 delay and cytotoxicity produced by cisplatin (CDDP) and camptothecin (CPT) in U251 glioma and DAOY medulloblastoma cells. Synchronized U251 were incubated with 20 microM CDDP in the presence or absence of 2 mM caffeine. DAOY cells were incubated with 100 nM CPT in the presence or absence of 2 nM staurosporine. Caffeine and staurosporine attenuated G2 delay produced by CDDP and CPT, respectively. Clonogenic assays indicated that continuous exposure to 2 mM caffeine substantially lowered the ID50 and ID90 of CDDP in U251 cells without significantly altering plating efficiency. Twenty-four-hour exposure to 2 nM staurosporine lowered the ID50 and ID90 of CPT in DAOY cells without significantly altering plating efficiency. Evaluation of programmed cell death using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assay indicated that one mechanism for synergistic cytotoxicty of caffeine with CDDP and staurosporine with CPT in U251 and DAOY cells, respectively, is to promote apoptosis. These results underscore the importance of understanding regulation of G2/M transition in brain tumor cells. Such an understanding may lead to novel therapies that target G2 check points to augment the efficacy of currently available treatments for brain tumors.

Apoptosis↗
PubMed1998

Inhibiting Ras prenylation increases the radiosensitivity of human tumor cell lines with activating mutations of ras oncogenes.

The influence of activated ras oncogenes on the sensitivity of human tumor cells to killing by radiation has been an unresolved question in radiobiology. We have examined this question by measuring the radiation sensitivity of human tumor cell lines with oncogenic mutations in their H- or K-ras genes after treatment with prenyltransferase inhibitors that prevent the posttranslational modification of ras required for its activity. Using two measures of clonogenic survival, we have demonstrated radiosensitization in cell lines with oncogenic H-ras mutations or with oncogenic K-ras mutations when ras processing was inhibited by prenyltransferase inhibitor treatment. In contrast, the inhibition of ras processing in cell lines expressing wild-type ras had no effect on radiation-induced cell death. The prenyltransferase inhibitors themselves inhibited clonogenic survival in some cases, but this inhibition did not correlate with ras mutational status. Although treatment with prenyltransferase inhibitors and radiation resulted in a greater reduction of clonogenicity than either treatment alone in cells with wild-type ras, treatment with both agents had a synergistic effect on cell killing in tumor cells with ras mutations. Our results demonstrate that the inhibition of oncogenic ras activity in human tumor cells can reduce the radiation survival of these cells, suggesting that oncogenic ras can contribute to radiation resistance in human tumors. These results further demonstrate the potential of using prenyltransferase inhibitors in combination with radiotherapy in the treatment of human malignancies.

Blotting, Western↗
PubMed1998

Requirement for matrix metalloproteinase-9 (gelatinase B) expression in metastasis by murine prostate carcinoma.

Although a number of effective therapies are available for localized prostate cancer, metastatic prostate cancer is difficult to treat and impossible to cure. Identification of the gene products that enable a prostatic carcinoma cell to metastasize should facilitate an understanding of the processes leading to metastasis. To characterize the contribution of matrix metalloproteinase-9 (MMP-9, gelatinase B or the 92-kd type IV gelatinase/collagenase) to the development of metastasis in prostate cancer, we reduced MMP-9 expression in metastatic murine prostatic carcinoma cells using a ribozyme. The ribozyme transfected cells had lower basal levels of MMP-9 as well as decreased levels after stimulation by transforming growth factor-beta or phorbol 12-myristate 13-acetate when compared with the parental cells or with control transfectants. The cells with down-regulated MMP-9 were unable to form lung colonies in the experimental metastasis assay, whereas the controls and parental cells readily formed metastases. All cell types readily formed tumors after injection and down-regulation of MMP-9 did not adversely affect the rate of tumor growth. Thus, MMP-9 expression is required for hematogenous metastasis in a murine prostate model system raising the possibility that it may play an equivalent role in human prostate cancer.

Animals↗