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Biomedical subjects

S Rockwell

Publications and source records attributed to S Rockwell.

At least 19 recordsLinked to original sources
PubMed2001

Reversal of mitomycin C resistance by overexpression of bioreductive enzymes in Chinese hamster ovary cells.

The clinical utility of antineoplastic agents is limited by the development of drug resistance by tumors. Mitomycin C (MC) is a bacterial product that must be enzymatically reduced to exert anticancer activity. We have demonstrated that expression of the bacterial MC resistance-associated (MCRA) protein in Chinese hamster ovary (CHO) cells confers profound resistance to this antibiotic under aerobic conditions, but not under hypoxia. MCRA produces resistance to MC by redox cycling of the activated hydroquinone intermediate back to the prodrug form. A CHO cell line developed by stepwise exposure to increasing concentrations of MC likewise expressed high level resistance to MC in air, but not under hypoxia. The overexpression of DT-diaphorase and NADPH:cytochrome c (P-450) reductase, two enzymes known to activate MC, restored sensitivity to MC in both MCRA-transfected and drug-selected cell lines. The level of sensitization was proportional to the quantity of enzyme activity expressed, supporting the concept that the levels of these two activating enzymes are important for sensitivity to MC. The findings of resistance to MC in air but not under hypoxic conditions and of restoration of sensitivity to MC by increasing levels of DT-diaphorase activity, properties not adequately explained by other resistance mechanisms (i.e., decreases in MC activation, repair of DNA lesions, and/or drug efflux), support the hypothesis that a functional mammalian homologue of MCRA may be involved in producing resistance to MC.

Animals↗
PubMed2001

Bioreductive metabolism of mitomycin C in EMT6 mouse mammary tumor cells: cytotoxic and non-cytotoxic pathways, leading to different types of DNA adducts. The effect of dicumarol.

The six DNA adducts formed in EMT6 mouse mammary tumor cells upon treatment with mitomycin C (MC) fall into two groups: (1) four guanine adducts of MC and (2) two guanine adducts derived from 2,7-diaminomitosene (2,7-DAM), the major reductive metabolite of MC. The two groups of adducts were proposed to originate from two pathways arising from reductive activation of MC: (a) direct alkylation of DNA and (b) formation of 2,7-DAM, which then alkylates DNA. The aim of this study was to test the validity of this proposal and to evaluate the significance of alkylation of DNA by 2,7-DAM. Treatment of the cells with 2,7-DAM itself yielded the same 2,7-DAM-guanine adducts as treatment with MC; however, 2,7-DAM was approximately 100-fold less cytotoxic than MC. The uptake and efflux of 2,7-DAM by EMT6 cells was comparable to that of MC, but 2,7-DAM alkylated DNA with higher efficiency than MC. These results validate the two proposed pathways and show that formation of 2,7-DAM-DNA adducts in MC-treated cells represents a relatively non-toxic pathway of reductive metabolism of MC. A selective stimulatory effect of dicumarol (DIC) on 2,7-DAM-DNA adduct formation in EMT6 cells treated with MC was also investigated. DIC had no effect on alkylation by MC in cell-free systems, nor did it have significant effects on adduct formation or cell survival for cells treated with 2,7-DAM. It is proposed that in the cell DIC stimulates a reductase enzyme located at subcellular sites where the activated MC species has no direct access to DNA and therefore is diverted into the non-cytotoxic pathway, which leads to the formation of 2,7-DAM and its adducts.

Animals↗
PubMed2001

Genomic instability in cancer.

Solid tumours have abnormal, deficient vascular and lymphatic systems. As a result, perfusion within these malignancies is inadequate and chaotic, and the cancers contain regions that are transiently and chronically exposed to low pH, severe hypoxia and nutrient deprivation. These microenvironmental inadequacies are present from the earliest point in the development of solid tumours, and are fully established while the neoplasms are still microscopic. Exposures to acidic and hypoxic environments have been shown to produce a wide range of cytogenetic changes. These include increases in mutation frequencies; deficits in DNA repair; DNA overreplication and gene amplification; the induction of chromosomal fragile sites, triggering genomic rearrangements; and changes in gene expression. Moreover, exposure of cells to adverse microenvironments such as those in solid tumours selects for cells which have defects in the structure or expression of the genes that normally regulate cell proliferation. The genetic changes and selection pressures induced by hypoxia may be critical in causing the development of the genomic instability and genetic heterogeneity which is characteristic of solid tumours and in fostering the evolution of relatively benign cell populations in solid tumours to increasingly malignant, increasingly aggressive phenotypes.

Cell Hypoxia↗
PubMed2001

Correlation between parental perception and actual childhood patterns of bicycle helmet use and riding practices: implications for designing injury prevention strategies.

BACKGROUND/PURPOSE: Bicycle injuries account for 10% of all pediatric traumatic deaths. Bicycle helmets have proven to decrease morbidity and mortality, yet trauma data show low helmet use among injured children. However, owning a bicycle helmet does not universally result in a child wearing a helmet. Furthermore, we hypothesize that parental perception of their children's use of the bicycle helmet may not reflect accurately true utilization by their child. To investigate this hypothesis the authors examined both parents' and their children's reports of bicycle ownership, supervision, riding patterns, and helmet use. METHODS: A random sample of grade 5 and 6 students (ages 8 to 12) and their parents were surveyed about bicycle ownership, riding patterns, supervision, and helmet use. The children and their guardians responded independently to the questionnaire. Statistical analysis was performed using the chi(2) test when indicated. RESULTS: Eighty-eight of 102 children (86%) responded. This represented 56% girls and 44% boys aged 8 to 12 years. Sixty-nine of 90 (77%) of the parents returned the survey. Ninety-six percent of the children owned a bicycle. A total of 87.5% of children owned a bicycle helmet. Eighty percent of the time children ride their bicycles on the road or sidewalk, with less then 20% on marked trails or parks. Parents reported that their children wear a helmet 90% of the time. In contrast, children report no helmet use in up to 61% of riding instances (P <.05). Parents themselves do not wear a helmet in greater then 60% when riding, which is correlated by their children. Seventy-one percent of the children report that they ride unsupervised the majority of the time. CONCLUSIONS: Bicycle and bicycle helmet ownership is high among this study group. There is a significant possibility that children will ride unsupervised, in at-risk situations, without wearing a helmet. Parental perceptions about bicycle helmet use by their children may not accurately reflect true utilization. In this study group parents appear as poor role models for their children. Injury prevention strategies need to focus on children and adults to improve effectiveness.

Accident Prevention↗
PubMed2001

The riddle of femininity: the interplay of primary femininity and the castration complex in analytic listening.

This study elucidates the application of an analytic attitude to questions of gender and sexuality. The paper reports on a study group's exploration of the relative heuristic use of two important organising concepts in analytic work with female analysands: primary feminity and the phallic castration complex. A tendency to cling to one position over the other skews analytic listening. Two cases are presented of women struggling to consolidate positive feminine identifications and, to that end, working through conflicting feminine identifications and defences against a resolution of the awareness of gender differences. Analytic listening requires a view of each psychic construction as a layer to be understood in its own right yet as a cloak soon to reveal the next layer--a different construction. The study includes observations on perverse fantasies in women.

Adult↗
PubMed2000

Pentoxifylline improves the oxygenation and radiation response of BA1112 rat rhabdomyosarcomas and EMT6 mouse mammary carcinomas.

Tumor hypoxia can significantly impact the efficacy of cancer therapy. Pentoxifylline, a methylxanthine derivative, can improve oxygen delivery to tissues and is widely used in the treatment of peripheral vascular disease and various cerebrovascular disorders. In this article, we show that pentoxifylline, combined with oxygen breathing, significantly improves the radiation response of two experimental tumors in vivo through improved tumor oxygenation. We also demonstrate that pentoxifylline does not directly radiosensitize EMT6 cells in vitro and does not modify the tumor radiation response when administered postirradiation to solid EMT6 tumors. Our findings confirm that preirradiation administration of pentoxifylline can improve radiation efficacy, but suggest that its role as a postirradiation modifier of treatment response, reported by others, may be tumor-specific.

Animals↗
PubMed2000

Interim results of a randomized trial of mitomycin C as an adjunct to radical radiotherapy in the treatment of locally advanced squamous-cell carcinoma of the cervix.

The purpose of this study was to determine the efficacy of mitomycin C as an adjunct to radiotherapy for the treatment of locally advanced cervix cancer. Patients with squamous-cell carcinoma of the cervix, stages IB2-IVA, were randomized to receive radiotherapy alone or radiotherapy with concomitant mitomycin C. An initial cohort of 160 patients, having a mean follow-up of 46 months, is analyzed. Intravenous mitomycin C, 15 mg/M(2), was given on the first and sixth week of radiotherapy. The 78 patients in the radiotherapy with mitomycin C group and 82 patients in the radiotherapy alone group have a comparable distribution by age and stage (mean age 47 years; stage IB 3%, IIA 11%, IIB 48%, IIIA 1%, IIIB 36%, IVA 3%). The four-year actuarial survival rates for radiotherapy with mitomycin C and radiotherapy alone were 72% and 56%, respectively (P = 0.13). The four-year actuarial disease-free survival rates for radiotherapy with mitomycin C and radiotherapy alone were 71% and 44%, respectively, a statistically significant difference (P = 0.01). The four-year actuarial local recurrence-free survival rates for patients receiving radiotherapy with mitomycin C and radiotherapy alone were 78% and 63%, respectively (P = 0.11). Differences in four-year distant recurrence-free survival between radiotherapy plus mitomycin C and radiotherapy alone were significantly different at 85% vs. 61% (P = 0.01); this analysis is not adjusted for local failure. On subgroup analysis, stage III-IVA patients had a four-year actuarial disease-free survival of 75% for radiotherapy plus mitomycin C compared with 35% for radiotherapy alone (P = 0.03). There were no treatment- related deaths. Mild hematologic toxicity was seen only in the group treated with mitomycin C. No excess in non-hematologic toxicity has been observed thus far with combined mitomycin C and radiotherapy. In this open phase III trial of mitomycin C as an adjunct to radical radiotherapy for squamous-cell carcinoma of the cervix, there were minimal hematologic effects and no increase in acute radiation reactions. A statistically significant difference in favor of patients receiving mitomycin C is shown for disease-free survival. Thus far, there are trends in favor of those patients receiving mitomycin C for survival and local control. Patients with more advanced stage disease, predominantly stage IIIB, appear to have the most benefit. These preliminary results support the hypothesis that targeting hypoxic cells may lead to a therapeutic enhancement in the radiotherapy of cervix cancer. This trial continues to accrue patients and follow-up data. Int. J. Cancer (Radiat. Oncol. Invest.) 90, 206-223 (2000).

Actuarial Analysis↗
PubMed2000

Diminished DNA repair and elevated mutagenesis in mammalian cells exposed to hypoxia and low pH.

The tumor microenvironment is characterized by regions of fluctuating and chronic hypoxia, low pH, and nutrient deprivation. It has been proposed that this unique tissue environment itself may constitute a major cause of the genetic instability seen in cancer. To investigate possible mechanisms by which the tumor microenvironment might contribute to genetic instability, we asked whether the conditions found in solid tumors could influence cellular repair of DNA damage. Using an assay for repair based on host cell reactivation of UV-damaged plasmid DNA, cells exposed to hypoxia and low pH were found to have a diminished capacity for DNA repair compared with control cells grown under standard culture conditions. In addition, cells cultured under hypoxia at pH 6.5 immediately after UV irradiation had elevated levels of induced mutagenesis compared with those maintained in standard growth conditions. Taken together, the results suggest that cellular repair functions may be impaired under the conditions of the tumor microenvironment, causing hypermutability to DNA damage. This alteration in repair capacity may constitute an important mechanism underlying the genetic instability of cancer cells in vivo.

Animals↗
PubMed2000

Antitumour effects of genetically engineered Salmonella in combination with radiation.

The antitumour efficacy of lipid A mutant Salmonella was evaluated alone and in combination with X-rays in mice bearing B16F10 or Cloudman S91 melanomas. Each treatment alone slowed tumour growth and prolonged survival, and the combined treatments produced supra-additive antitumour effects. That is, in dose-response studies with single doses of Salmonella and increasing doses of radiation, the two agents together caused suppression of tumour growth that was greater than that calculated for additivity. The results suggest that the combination of these genetically engineered Salmonella with radiotherapy could be a new and beneficial treatment for solid tumours.

Animals↗
PubMed2000

Structure of adduct X, the last unknown of the six major DNA adducts of mitomycin C formed in EMT6 mouse mammary tumor cells.

Treatment of EMT6 mouse mammary tumor cells with mitomycin C (MC) results in the formation of six major MC-DNA adducts. We identified the last unknown of these ("adduct X") as a guanine N(2) adduct of 2, 7-diaminomitosene (2,7-DAM), in which the mitosene is linked at its C-10 position to guanine N(2). The assigned structure is based on UV and mass spectra of adduct X isolated directly from the cells, as well as on its difference UV, second-derivative UV, and circular dichroism spectra, synthesis from [8-(3)H]deoxyguanosine, and observation of its heat stability. These tests were carried out using 17 microg of synthetic material altogether. The mechanism of formation of adduct X involves reductive metabolism of MC to 2,7-DAM, which undergoes a second round of reductive activation to alkylate DNA, yielding adduct X and another 2,7-DAM-guanine adduct (adduct Y), which is linked at guanine N7 to the mitosene. Adduct Y has been described previously. Adduct X is formed preferentially at GpC, while adduct Y favors the GpG sequence. In contrast to MC-DNA adducts, the 2,7-DAM-DNA adducts are not cytotoxic.

Animals↗
PubMed2000

Biological basis of radiation sensitivity. Part 1: Factors governing radiation tolerance.

Local tumor recurrence after radiation therapy is due primarily to the failure to eradicate all of the tumor cells within the treatment fields. Theoretically, all cancers could be controlled locally if a sufficiently high radiation dose could be delivered to a treatment volume that encompassed all of the tumor cells. In practice, however, the administration of a radiation dose high enough to sterilize all of the tumor cells would pose an unacceptably high risk of severe damage to normal tissues. Technologic improvements in the delivery of therapeutic radiation have led to some improvements in the therapeutic ratio (i.e., the ratio of the dose required to eradicate every tumor cell to the dose that produces unacceptable normal tissue toxicity). Further significant improvements in the therapeutic ratio will drive from an understanding of the mechanisms governing the sensitivity of malignant and normal cells to radiation. Part 1 of this two-part article reviews the clinical and tissue kinetic factors that govern the sensitivity of normal tissues and organs to ionizing radiation. Part 2, which will appear in next month's issue, describes recent insights into the cellular and molecular pathways that determine the sensitivity of normal cells and tumor cells to radiation.

Antineoplastic Agents↗
PubMed2000

Biological basis of radiation sensitivity. Part 2: Cellular and molecular determinants of radiosensitivity.

Recent studies have elucidated some of the molecular and cellular mechanisms that determine the sensitivity or resistance to ionizing radiation. These findings ultimately may be useful in devising new strategies to improve the therapeutic ratio in cancer treatment. Despite the rapid advances in knowledge of cellular functions that affect radiosensitivity, we still cannot account for most of the clinically observed heterogeneity of normal tissue and tumor responses to radiotherapy, nor can we accurately predict which individual tumors will be controlled locally and which patients will develop more severe normal tissue damage after radiotherapy. However, several candidate genes for which deletion or loss of function mutations may be associated with altered cellular radiosensitivity (e.g., ATM, p53, BRCA1, BRCA2, DNA-PK) have been identified. Some of the differences in normal tissue sensitivity to radiation may stem from mutations with milder effects, heterozygosity, or polymorphisms of these genes. Finally, molecular mechanisms linking genetic instability, radiosensitivity, and predisposition to cancer are being unraveled.

Apoptosis↗
PubMed1999

Mitomycin resistance in mammalian cells expressing the bacterial mitomycin C resistance protein MCRA.

The mitomycin C-resistance gene, mcrA, of Streptomyces lavendulae produces MCRA, a protein that protects this microorganism from its own antibiotic, the antitumor drug mitomycin C. Expression of the bacterial mcrA gene in mammalian Chinese hamster ovary cells causes profound resistance to mitomycin C and to its structurally related analog porfiromycin under aerobic conditions but produces little change in drug sensitivity under hypoxia. The mitomycins are prodrugs that are enzymatically reduced and activated intracellularly, producing cytotoxic semiquinone anion radical and hydroquinone reduction intermediates. In vitro, MCRA protects DNA from cross-linking by the hydroquinone reduction intermediate of these mitomycins by oxidizing the hydroquinone back to the parent molecule; thus, MCRA acts as a hydroquinone oxidase. These findings suggest potential therapeutic applications for MCRA in the treatment of cancer with the mitomycins and imply that intrinsic or selected mitomycin C resistance in mammalian cells may not be due solely to decreased bioactivation, as has been hypothesized previously, but instead could involve an MCRA-like mechanism.

Aerobiosis↗
PubMed1999

Hypoxia-selective nitrobenzyloxycarbonyl derivatives of 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazines.

Some 4- and 2-(nitrobenzyloxycarbonyl)-1, 2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazines (4, 6, and 7) were synthesized and evaluated for their ability to exert preferential toxicity to hypoxic EMT6 mammary carcinoma cells using a colony-forming assay. Of these, the 4,5-dimethoxy-2-nitro analogue 6 (50 microM, 1-h exposure) caused greater than 3 logs of kill of hypoxic cells, with relatively minor toxicity to corresponding aerobic cells. The ability of 4-nitro (4) and 4,5-dimethoxy-2-nitro (6) analogues to reach and kill hypoxic cells of solid tumors was also demonstrated using intradermally implanted EMT6 solid tumors in mice. In addition, a possible source of toxicity to normal tissue, i. e., the activation of the 4-nitrobenzyl derivative 4 by glutathione S-transferase-catalyzed thiolysis, was essentially eliminated by replacing one of the benzylic methylene protons by a methyl group. The 4-nitro (4) and 4,5-dimethoxy-2-nitro (6) analogues also appear to be reduced more easily under acidic conditions (pH 6.0) than under neutral conditions, as measured by differential pulse polarography. Since the pH in hypoxic regions is often lower than that in adjacent aerobic regions, this property should aid in the cytotoxic action of these agents against hypoxic cells of solid tumors.

Animals↗
PubMed1999

The molecular and cellular basis of radiosensitivity: implications for understanding how normal tissues and tumors respond to therapeutic radiation.

We have provided an overview of recent studies that have greatly expanded our knowledge of the molecular and cellular mechanisms that determine the sensitivity or resistance to ionizing radiation. Much of this knowledge was obtained by studying tumor and nontumor cell types that under- or overexpress proteins involved in the regulation of the DNA damage response, cell cycle progression, growth factor signal transduction, and apoptosis. These findings may ultimately be useful in devising new strategies to improve the therapeutic ratio in cancer treatment. Despite the rapid advances in knowledge of cellular functions that affect radiosensitivity, we still cannot account for most of the clinically observed heterogeneity of normal tissue and tumor responses to radiotherapy; nor can we accurately predict which individual tumors will be locally controlled and which patients will develop more severe normal tissue damage after radiotherapy. However, several candidate genes for which deletion or loss of function mutations may be associated with altered cellular radiosensitivity (e.g., ATM, p53, BRCA2) have been identified. Some of the differences in normal tissue sensitivity to radiation may occur because of mutations with milder effects, heterozygosity, or polymorphisms of these genes. Finally, molecular mechanisms linking genetic instability, radiosensitivity, and predisposition to cancer are being examined.

Acute Disease↗
PubMed1998

Scatter factor protects epithelial and carcinoma cells against apoptosis induced by DNA-damaging agents.

Scatter factor (SF) (hepatocyte growth factor) is a cytokine that may play a role in human breast cancer invasiveness and angiogenesis. We now report that SF can block the induction of apoptosis by various DNA damaging-agents, including cytotoxic agents used in breast cancer therapy. SF protected MDA-MB-453 human breast cancer cells, EMT6 mouse mammary tumor cells and MDCK renal epithelial cells against apoptosis induced by adriamycin (ADR), X-rays, ultraviolet radiation, and other agents. Protection was observed in assays of DNA fragmentation, cell viability (MTT), and clonogenic survival. Protection of MDA-MB-453 cells against ADR was dose- and time-dependent; maximal protection required pre-incubation with 75-100 ng/ml of SF for 48 h or more. Protection required functional SF receptor (c-Met), but was not dependent on p53. Western blotting analysis revealed that pre-treatment of MDA-MB-453 cells with SF inhibited the ADR-induced decreases in the levels of Bcl-XL, an anti-apoptotic protein related to Bcl-2; and the dose-response and time course characteristics for SF-mediated increases in the Bcl-XL protein levels of ADR-treated cells were consistent with the degrees of protection against apoptosis observed under the same conditions. Furthermore, Bcl-XL levels were not down-regulated by ADR in MDA-MB-231 breast cancer cells, consistent with the finding that SF failed to protect these cells against ADR, despite the fact that they contain functional c-Met receptor. In contrast to Bcl-XL, SF blocked ADR-induced increases in c-Myc and inhibited the expression of p21WAF1/CIP1 and of the BRCA1 protein in MDA-MB-453 cells. However, SF did not cause significant changes in the cell cycle distribution of ADR-treated cells. These findings suggest that SF-mediated protection of human breast cancer cells may involve inhibition of one or more pathways required for the activation of apoptosis and may particularly target the anti-apoptotic mitochondrial membrane pore-forming protein Bcl-XL as a component of the protective mechanism. By implication, the accumulation of SF within human breast cancers may contribute to the development of a radio- or chemoresistant phenotype.

Animals↗
PubMed1998

The intracellular location of NADH:cytochrome b5 reductase modulates the cytotoxicity of the mitomycins to Chinese hamster ovary cells.

NADH:cytochrome b5 reductase activates the mitomycins to alkylating intermediates in vitro. To investigate the intracellular role of this enzyme in mitomycin bioactivation, Chinese hamster ovary cell transfectants overexpressing rat NADH:cytochrome b5 reductase were generated. An NADH:cytochrome b5 reductase-transfected clone expressed 9-fold more enzyme than did parental cells; the levels of other mitomycin-activating oxidoreductases were unchanged. Although this enzyme activates the mitomycins in vitro, its overexpression in living cells caused decreases in sensitivity to mitomycin C in air and decreases in sensitivity to porfiromycin under both air and hypoxia. Mitomycin C cytotoxicity under hypoxia was similar to parental cells. Because NADH:cytochrome b5 reductase resides predominantly in the mitochondria of these cells, this enzyme may sequester these drugs in this compartment, thereby decreasing nuclear DNA alkylations and reducing cytotoxicity. A cytosolic form of NADH:cytochrome b5 reductase was generated. Transfectants expressing the cytosolic enzyme were restored to parental line sensitivity to both mitomycin C and porfiromycin in air with marked increases in drug sensitivity under hypoxia. The results implicate NADH:cytochrome b5 reductase in the differential bioactivation of the mitomycins and indicate that the subcellular site of drug activation can have complex effects on drug cytotoxicity.

Animals↗