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R E Meyn

Publications and source records attributed to R E Meyn.

At least 37 records · Page 2Linked to original sources
PubMed1999

Analysis of redox regulation of cytochrome c-induced apoptosis in a cell-free system.

In this study, we investigated the importance of redox and Bcl-2 status on cytochrome c-mediated apoptosis. Two mouse lymphoma cell lines, LYas and LYar that express Bcl-2 protein at different levels, were used to reconstitute a cell-free system. Cytoplasmic extracts made from apoptosis-sensitive LYas cells 2.5 h after exposure to 5 Gy gamma-radiation were able to induce apoptosis in isolated nuclei, whereas extracts made from LYas cells at time points earlier than 2. 5 h, or from Bcl-2-overexpressing, apoptosis-resistant LYar cells at all time points after irradiation were inactive. Apoptotic activity was restored to inactive extracts by the addition of oxidized but not reduced cytochrome c. Cytochrome c reductase was able to inhibit apoptosis in extracts made from LYas cells 2.5 h after irradiation and LYar extracts activated by addition of oxidized cytochrome c. Antioxidants, but not oxidant defensive enzymes, blocked apoptosis implying that antioxidants might alter the redox state of factors important in mediating apoptosis. These findings confirm the importance of cellular redox state during apoptosis and are consistent with a role for Bcl-2 in regulating this redox state.

Animals↗
PubMed1999

Interactions of apoptosis, proliferation and host age in the regression of the mouse mammary preneoplasia, TM3, carrying an unusual mutation in p53.

We have developed an in vivo model system of mouse mammary preneoplasias in order to examine the cell and molecular changes that occur during tumorigenesis. Most of these preneoplasias are characterized by an alveolar hyperplasia morphologically similar to that present in normal pregnant mammary gland, but have tumor forming capabilities ranging from very low to high. One of these hyperplasias, the TM3 HOG (transformed mammary hyperplastic outgrowth), forms tumors infrequently and has the unusual characteristic of spontaneous regression. We have observed that 7-8 months post-transplantation into the cleared mammary fat pad of a BALB/c mouse, the TM3 hyperplasia will begin to regress, leaving only a sparse ductal tree with remnant alveolar structures by 10-12 months post-transplantation. We have sought to elucidate the mechanism of this regression by determining the apoptotic and proliferative rates of the alveolar cells during TM3 HOG development. Studies show that apoptotic rates in the TM3 HOG are consistently high (4-7%) at all times after transplantation. This apoptotic rate is higher than the rates found in other preneoplasias in our system and approach the rates observed in the normal involuting gland. An unusual p53 mutation, a serine insertion at codon 233, may be causally related to the high spontaneous apoptotic frequencies as well as elevated inducible apoptotic frequencies in TM3. In addition, a sudden decrease ( approximately 63%) in proliferation occurs around 8 months post-transplantation. Furthermore, transplantation experiments indicate that the ability of the 8-month-old host and/or mammary gland to support growth of preneoplastic mammary tissues is markedly diminished compared with 3- or 6-month-old hosts. The results presented here suggest that the persistent high apoptotic rates, concomitant with decreased proliferation rates, may be responsible for TM3's regression and implicate a unique mutant p53 as a causal factor. Additionally, the results suggest that host determinants can interact with specific molecular changes in the preneoplastic cells to influence growth and progression of the preneoplastic populations.

Age Factors↗
PubMed1999

p53 tumor suppressor gene therapy for cancer.

Gene therapy has the potential to provide cancer treatments based on novel mechanisms of action with potentially low toxicities. This therapy may provide more effective control of locoregional recurrence in diseases like non-small-cell lung cancer (NSCLC) as well as systemic control of micrometastases. Despite current limitations, retroviral and adenoviral vectors can, in certain circumstances, provide an effective means of delivering therapeutic genes to tumor cells. Although multiple genes are involved in carcinogenesis, mutations of the p53 gene are the most frequent abnormality identified in human tumors. Preclinical studies both in vitro and in vivo have shown that restoring p53 function can induce apoptosis in cancer cells. High levels of p53 expression and DNA-damaging agents like cisplatin (Platinol) and ionizing radiation work synergistically to induce apoptosis in cancer cells. Phase I clinical trials now show that p53 gene replacement therapy using both retroviral and adenoviral vectors is feasible and safe. In addition, p53 gene replacement therapy induces tumor regression in patients with advanced NSCLC and in those with recurrent head and neck cancer. This article describes various gene therapy strategies under investigation, reviews preclinical data that provide a rationale for the gene replacement approach, and discusses the clinical trial data available to date.

Animals↗
PubMed1998

ASTRO research fellowship: the role of BCL-2 and glutathione in an antioxidant pathway to prevent radiation-induced apoptosis.

PURPOSE: The expression of the bcl-2 proto-oncogene has been associated with resistance to radiation-induced apoptosis. There is evidence that the bcl-2 protein acts in an antioxidant pathway to block the effects of reactive oxygen species that mediate apoptosis possibly by increasing the levels of intracellular glutathione. Our hypothesis is that pretreatment of radiation-sensitive cells, known to lack bcl-2 expression, with antioxidants will reduce radiation-induced apoptosis. For this purpose, the apoptotic response to radiation and the intracellular levels of GSH were tested before and after pretreatment with antioxidants in two murine lymphoma cell lines, a radiation-resistant, bcl-2- expressing (LY-ar) line and a radiation-sensitive, non-bcl-2-expressing (LY-as) line. METHODS AND MATERIALS: LY-ar and LY-as cells were irradiated at 0,1,2,3, and 4 hours before collection. The intracellular levels of reduced (GSH) and oxidized (GSSG) glutathione were determined by the use of the fluorescent dye o-phthalaldehyde. LY-as cells were treated with GSH ethyl-ester for 1 and 2 hours after irradiation. Apoptotic response was measured by the DNA fragmentation assay. The radiation dose was 2.5 Gy. RESULTS: After irradiation, the apoptotic rate of LY-ar and LY-as cells was 10-20% and 50-70% respectively. LY-ar cells had higher intracellular GSH and GSSG levels compared to LY-as cells by 69.9% and 91.9% respectively and the GSH/GSSG ratio in LY-ar and LY-as cells was 15.09 and 17.09 respectively. GSH levels did not change during the first 2 hours after irradiation; however, there was a 49% and 84% reduction at 3 and 4 hours after irradiation, respectively, times at which the LY-as cells have already fragmented their DNA. Treatment of LY-as cells with GSH ethyl-ester at a concentration of 7 mM for 1 and 2 hours resulted in 70% and 231% increases in the intracellular GSH levels respectively. Treatment of LY-as cells with GSH ethyl-ester for 1 and 2 hours also conferred a 25-50% decrease in their apoptotic response after irradiation. CONCLUSIONS: GSH and GSSG levels are elevated in radiation-resistant, bcl-2-expressing murine lymphoma cells compared to radiation-sensitive, non-bcl-2-expressing cells. GSH levels decline only in radiation-sensitive cells after irradiation but this appears to occur at the time of apoptotic cell death. Exogenous thiols increase intracellular GSH levels and repress radiation-induced apoptosis. In conclusion, intracellular thiols appear to be involved in protecting cells from apoptotic cell death. Further investigation should be directed in identifying substances which by lowering intracellular thiols may result in sensitization to radiation.

Animals↗
PubMed1998

Bcl-2 expression causes redistribution of glutathione to the nucleus.

In this study we used HeLa cells transfected with a conditional Bcl-2 expression construct to study the effects of Bcl-2 on reduced glutathione (GSH) metabolism. Our previous work demonstrated that depletion of GSH by culturing cells in tissue culture medium lacking the amino acids cysteine and methionine, essential for GSH biosynthesis, caused cells overexpressing Bcl-2 to become sensitized to apoptotic induction. Here we report that Bcl-2 also dramatically alters GSH compartmentalization. Cellular distribution of GSH, assayed by confocal microscopy, revealed that when Bcl-2 expression was suppressed GSH was uniformly distributed primarily in the cytosol, whereas overexpression of Bcl-2 led to a relocalization of GSH into the nucleus. Isolated nuclei readily accumulated radiolabeled GSH and maintained higher nuclear GSH concentration in direct relation to Bcl-2 nuclear protein levels. Moreover, exogenous GSH blocked apoptotic changes and caspase activity in isolated nuclei exposed to the pro-apoptotic protease granzyme B. Our results indicate that one of the functions of Bcl-2 is to promote sequestration of GSH into the nucleus, thereby altering nuclear redox and blocking caspase activity as well as other nuclear alterations characteristic of apoptosis. We speculate that this mechanism contributes to the suppression of apoptosis in cells with elevated Bcl-2 levels.

Animals↗
PubMed1998

Radiation-induced tumorigenesis in preneoplastic mouse mammary glands in vivo: significance of p53 status and apoptosis.

In mouse mammary tumorigenesis, p53 mutations facilitate tumorigenesis in concert with other oncogenic alterations. Ionizing radiation enhances tumorigenesis in preneoplastic mammary outgrowth lines and induces p53-dependent apoptosis. We asked if normal p53 function modulates radiation-induced tumorigenesis in preneoplastic mammary lesions by affecting the apoptotic pathway of cell deletion. Three different hyperplastic outgrowth lines were compared. Outgrowth line D1 overexpressed wild-type p53 and responded to irradiation with enhanced tumorigenicity but no induction of apoptosis. Outgrowth line TM12 exhibited normal wild-type p53 expression and responded to irradiation with no alteration in tumorigenicity but with a marked increase in apoptosis. Outgrowth line TM2L also exhibited normal wild-type p53 expression and responded to irradiation with a marked enhancement in both tumorigenicity and apoptosis. These results indicate that the two radiation-induced responses, apoptosis and tumorigenesis, are dissociable events in the mammary gland. Furthermore, radiation-induced tumorigenicity was not abrogated by either enhanced wild-type p53 expression or a robust apoptotic response. The radiation dose of 5 Gy most likely induces multiple genetic alterations in surviving cells, including genomic instability, and this may account for the tumorigenicity. Future experiments will examine lower doses of irradiation that still induce a significant apoptotic response but significantly less genomic instability.

Animals↗
PubMed1998

Reversing drug resistance in bcl-2-expressing tumor cells by depleting glutathione.

The immense research effort in cancer cell physiology has led to an appreciation of the molecular and biochemical pathways that regulate cellular responses to endogenous and exogenous insults. Similarly, in tumor cells, there are multiple overlapping pathways that, once activated, impart resistance to therapeutic intervention. The multi drug resistance pathway is one such pathway. In this review, we will present current ideas concerning a mechanism of tumor cell resistance that involves the inability to undergo apoptosis. The bcl-2 family of proteins are known to regulate apoptosis in response to a wide variety of toxic agents. Additionally, recent evidence points to bcl-2 involvement in the regulation of antioxidant pathways mediated by glutathione. This new information will be discussed in some detail and strategies for overcoming these resistance mechanisms that may have clinical utility will be presented.

Journal Article↗
PubMed1998

Bcl-2 expression correlates with apoptosis induction but not loss of clonogenic survival in small cell lung cancer cell lines treated with etoposide.

The influence of bcl-2 oncogene expression on etoposide-induced apoptosis and clonogenic survival was investigated in five small cell lung cancer (SCLC) cell lines, three of which were bcl-2-expressing and two of which were non-bcl-2-expressing. The bcl-2-expressing lines displayed a lower apoptosis propensity than the non-bcl-2-expressing lines. When bcl-2-expressing cells were incubated in cystine/ methionine-free (CMF) medium, etoposide-induced apoptosis was restored to levels comparable to those seen in non-bcl-2-expressing lines. However, the endpoint of clonogenic survival after drug treatment did not display any consistent pattern that correlated with bcl-2 status. In addition, treatment of the two bcl-2-expressing cell lines with etoposide in CMF medium did not modify their clonogenic survival curves compared to treatment in regular medium. These results are consistent with the idea that bcl-2 expression modulates etoposide-induced apoptosis but not clonogenic survival.

Antineoplastic Agents, Phytogenic↗
PubMed1997

Resistance to radiation-induced apoptosis in Bcl-2-expressing cells is reversed by depleting cellular thiols.

The mechanism by which Bcl-2 oncogene expression inhibits radiation-induced apoptosis has been investigated in two mouse lymphoma cell lines: line LY-as is radiation sensitive, displays substantial radiaton-induced apoptosis, and expresses low levels of Bcl-2; line LY-ar is radiation-resistant, displays a low apoptosis propensity, and expresses 30-fold higher amount of Bcl-2 protein than does the sensitive line. We observed that upon incubation in cystine/methionine-free (C/M-) medium, radiation-induced apoptosis in the LY-ar cells was restored to levels comparable to that seen in the LY-as cells. lntracellular glutathione (GSH) concentrations in LY-ar cells incubated in C/M- medium plummeted to 50% of control values within 2 h. LY-ar cells treated with diethyl maleate (DEM) or diamide, agents that deplete cellular thiols, had increased susceptibility to radiation-induced apoptosis in a manner similar to C/M- medium. These results are consistent with the general idea that Bcl-2 expression blocks apoptosis through an antioxidant pathway that involves cellular thiols. That Bcl-2-expressing tumor cells can be sensitized by exogeneous agents that modify cellular thiols offers strategies for overcoming such resistance.

Animals↗
PubMed1997

Modulating Ca2+ in radiation-induced apoptosis suppresses DNA fragmentation but does not enhance clonogenic survival.

The role of intracellular Ca2+ in radiation-induced apoptosis was studied in a cell line derived from a mouse B-cell lymphoma (LY-TH). These cells had previously been shown to be sensitive to radiation and to die by apoptosis. The cell permeant Ca2+ chelator (acetyoxymethyl-)1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tet raacetic acid (BAPTA/AM) reduced the DNA fragmentation characteristic of apoptosis but had no effect on clonogenic survival. Intracellular Ca2+ concentrations measured using the fluorescent indicator fura-2 only slowly increased over control values after cells were irradiated unlike the rapid increase observed in other systems. Our results indicate that modulating the endpoint of DNA fragmentation using some agents may not necessarily alter the cells' commitment to death as determined by clonogenic survival assays. This suggests that such agents play a role downstream of early initiation steps in apoptosis and modulate only particular features of apoptosis after the cell is committed to die.

Animals↗
PubMed1997

Apoptosis and response to radiation: implications for radiation therapy.

Apoptosis is a mode of cell death that is currently of intense research interest in developmental and cancer biology. For more than 40 years, radiobiologists have been aware of cells in irradiated specimens that display the features of apoptosis. Despite this knowledge, however, it has proved difficult to elucidate the role of apoptosis in end points important in radiation therapy and to distinguish its role from that of other modes of cell death, namely, reproductive cell death. In this article, the evidence relevant to this question will be critically reviewed. Conclusions that have significant implications for radiation therapy will be drawn to suggest that tumor cells with apoptotic propensity are more sensitive to radiation. If this hypothesis is confirmed, strategies can be envisioned that may restore apoptotic propensity to radioresistant tumor cells for therapeutic benefit.

Adenocarcinoma↗
PubMed1996

Apoptosis and downstaging after preoperative radiotherapy for muscle-invasive bladder cancer.

PURPOSE: To determine the relationship between pretreatment apoptosis levels and clinical-to-pathologic downstaging resulting from preoperative radiotherapy. METHODS AND MATERIALS: Between 1960-1983, 338 patients were dispositioned to receive preoperative radiotherapy 4-6 weeks prior to radical cystectomy for muscle-invasive transitional cell carcinoma of the bladder. Of these, adequate hematoxylin and eosin stained tissue sections for morphologic analysis of apoptosis were available in 158 patients. These patients were treated to a median dose of 50 Gy at 2 Gy per fraction. Median follow-up was 90 months. The apoptotic index (AI) was calculated from the ratio of the number of apoptotic cells divided by the total counted and multiplied by 100. A minimum of 500 cells were counted from each patient. RESULTS: The average AI for the whole group (n = 158) was 2.0 +/- 1.3 (+/- SD), with a median of 1.8. The association of AI to clinical stage was significant with AI averages of 1.8 for Stage T2 (n = 56), 1.9 for T3a (n = 51), and 2.4 for T3b (p = 0.038, Kendall Correlation). The relationship of AI to radiotherapy response also was significant with an average of 2.2 for those who were downstaged (n = 103), 1.9 for those in whom the stage remained unchanged (n = 20), and 1.7 for those who were upstaged (n = 35, p = 0.054, Kendall Correlation). The other significant correlations with AI were for the factors, grade, mitotic index, number of tumors, and gender. The AI was then categorized into three groups ( < or = 1, > 1, and < or = 3, and > 3) to examine the prognostic significance of this parameter. The distributions of patients by clinical stage, grade, mitotic index, number of tumors, radiotherapy response, and hemoglobin level were significantly associated with AI using this grouping. When the analysis of the distribution of patients by radiation response and AI was segregated by stage, a significant correlation was observed only for those with Stage T3b disease (p = 0.006); 93% of T3b patients with an AI > 3 were downstaged, while in 7% the stage remained unchanged and none were upstaged. The relationship of AI to 5-year actuarial patient outcome was investigated using several end points and although no significant correlations were observed, a trend was seen for improved survival when AI was > 3 (71% vs. 41%, p = 0.09) for Stage T3b patients. CONCLUSION: The AI correlated most strongly with radiotherapy response for patients with clinical stage T3b disease, the one subgroup of patients wherein preoperative radiotherapy is likely to be of the most benefit. Further investigation of pretreatment apoptosis levels as a marker of anticancer response is needed, especially for patients treated with chemotherapy and radiotherapy with the goal of bladder preservation.

Aged↗
PubMed1996

Radiation-induced apoptosis in normal and pre-neoplastic mammary glands in vivo: significance of gland differentiation and p53 status.

The tumor suppressor gene p53 maintains the integrity of the genome by stimulating apoptosis in cells that have sustained DNA damage. The p53 gene is frequently altered in human cancers, including breast cancer, and such alterations are thought to result in genomic instability and aneuploidy, 2 hallmarks of anaplastic cells. We used radiation as a DNA-damaging agent to test the role of p53 in controlling apoptosis propensity in pre-neoplastic mammary lesions in mice. Four different pre-neoplastic mammary outgrowth lines, D1, TM2H, TM4 and TM12, were maintained by serial transplantation in the cleared mammary fat pads of syngeneic BALB/c mice. These lines have known alterations in p53 expression: TM12 has normal expression, D1 over-produces wild-type protein, TM2H contains a deletion resulting in a null phenotype and TM4 produces a mutant protein. Mice bearing the various outgrowths were irradiated with 5 Gy, and apoptosis was scored by examination of histological sections prepared from the outgrowths 6 hr after irradiation. The TM12 outgrowths, but not the TM2H outgrowths, exhibited radiation-induced apoptosis. The D1 and TM4 lines expressed radiation-induced apoptosis while the fat pads were being repopulated; however, the induced apoptosis declined to low levels as the mice aged. These results are consistent with the hypothesis that normal p53 function is important if mammary cells with DNA damage are to be deleted by apoptosis.

Animals↗
PubMed1996

Programmed cell death and radioresistance.

Whereas apoptosis is a critical mode of cell deletion in normal organism development, apoptotic cells are also observed in tumors, especially following cytotoxic treatments, leading to questions about their role in tumor response to therapy. We have conducted a series of studies using murine tumor models and found that the ability of the tumor cells to undergo apoptosis correlates with tumor response to radiation. The best correlation was with the pretreatment apoptotic index, suggesting that apoptosis in some tumors may govern radiocurability by regulating the number of tumor clonogens. However, other roles for apoptosis in tumor response to radiation have not been ruled out. One of the important observations that has come from this work has been the heterogeneity in apoptosis propensity both within the cell population of a given tumor and among different types of tumors. Such findings underscore the fact that apoptosis is under complex genetic control and that some of the same oncogenes and tumor suppressor genes that are responsible for tumor initiation and progression to malignancy also dictate the apoptotic response to treatment. Understanding the biochemical and molecular pathways that govern this process may ultimately allow the development of strategies for modulating apoptosis for therapeutic benefit.

Animals↗
PubMed1996

Adenoviral-mediated wild-type p53 gene expression sensitizes colorectal cancer cells to ionizing radiation.

Wild-type p53 gene transfer into the SW620 colorectal carcinoma cell line was performed using the replication-defective adenovirus Ad5/CMV/p53 to evaluate the effect of wild-type p53 expression on radiation sensitivity. The results indicated that infection with Ad5/CMV/p53 sensitized the cells. The survival at 2 Gy was reduced from 55 to 23%. Flow cytometric analysis of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay-labeled cells and in situ TUNEL staining of xenograft tumors demonstrated an increase in labeled cells with combination treatment, indicating increased apoptosis in cells treated with Ad5/CMV/p53 before irradiation. A significant enhancement of tumor growth suppression by this combination strategy was observed in a s. c. tumor animal model compared to p53 gene therapy alone. The delay in regrowth to control tumor size of 1000 mm3 was 2 days for 5 Gy, 15 days for Ad5/CMV/p53, and 37 days for Ad5/CMV/p53 + 5 Gy, indicating synergistic interactions. These data indicate that the delivery of wild-type p53 to cells with p53 mutations increases their radiation sensitivity, and this may be accomplished by adenoviral-mediated gene therapy.

Adenoviridae↗
PubMed1995

Radiation-induced apoptosis: relevance to radiotherapy.

Radiation-induced apoptosis is reviewed in terms of: (a) the identification of apoptotic and necrotic cells, (b) observations in vitro and in vivo of radiation-induced apoptosis, (c) genes controlling apoptosis, (d) evidence that the target may be the plasma membrane or nuclear DNA, (e) quantitative comparisons of apoptotic death and reproductive (clonogenic) death, (f) the importance of radiation-induced apoptosis in radiotherapy, and (g) studies of radiation-induced apoptosis that are needed. High priority should be placed on determining the molecular pathways that are important in the expression and modulation of radiation-induced apoptosis. Specifically, the events that modulate the apoptosis that occurs in interphase before the cell can divide should be distinguished from the events before division that modulate the misrepair of DNA damage, that results in chromosomal aberrations observed in mitotic cells, which in turn cause the progeny of the dividing cell with aberrations to die by either apoptosis or necrosis. Then, molecular events that determine whether a cell that divides with or without a chromosomal aberration will produce progeny that apoptose or necrose need to be identified. These considerations are important for determining how modulation of radiation-induced apoptosis will affect the ultimate clonogenic survival, and possibly genomic instability in the surviving progeny.

Animals↗
PubMed1995

ASTRO Research Fellowship: apoptosis as a predictor of tumor response to radiation in stage IB cervical carcinoma. American Society for Therapeutic Radiology and Oncology.

PURPOSE: Levels of apoptosis predict for tumor responsiveness to radiation in various animal systems. To investigate the potential role of apoptosis as a predictor of response in human tumors, a retrospective review was undertaken of patients with adenocarcinoma of the cervix whose primary lesion at presentation measured at least 4 cm and who underwent definitive radiation therapy. A previous report had indicated that roughly half this group of patients should have a long-term relapse free survival. METHODS AND MATERIALS: Pretreatment biopsy specimens of 44 patients with Stage IB adenocarcinoma of the cervix, whose primary lesion at presentation measured at least 4 cm in greatest dimension, were scored for apoptosis by two independent investigators without knowledge of the treatment outcome, and the results were averaged. Actuarial methods were used to assess overall survival, disease-free survival, determinate survival, and local control as a function of the baseline level of apoptosis. Patients ranged in age from 21 to 87 years and were treated with definitive radiotherapy between 1964 and 1989. Follow-up for the surviving patients ranged from 1 to 278 months, with a mean of 101 months. RESULTS: Patients whose tumors had a baseline level of apoptosis above the median value (2%) had a better overall survival than those with lower levels of apoptosis (p = 0.056). A similar trend for disease-free survival (p = 0.32) and determinate survival (p = 0.27) did not reach statistical significance, perhaps because of the small number of patients. Because only 6 of the 44 patients (13%) had a local tumor failure, it was not possible to establish a correlation between the pretreatment level of apoptosis and the local tumor control by radiation. CONCLUSION: The baseline level of apoptosis predicted for survival in patients with Stage IB cervical adenocarcinoma. Further investigation of the measurement of apoptosis as a potential predictive assay is warranted in other human tumor systems.

Adenocarcinoma↗