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M N Gould

Publications and source records attributed to M N Gould.

At least 37 records · Page 2Linked to original sources
PubMed1998

Harvey ras results in a higher frequency of mammary carcinomas than Kirsten ras after direct retroviral transfer into the rat mammary gland.

Exclusive activation of either the Harvey-, Kirsten-, or N-ras gene is often found in human and rodent cancers, although the mechanisms responsible for tissue-specific ras gene activation are poorly understood. In this study, the contribution of ras gene expression and Ras protein activity to the tissue-specificity of ras gene activation was investigated using the rat mammary carcinogenesis model where ras activation, when it occurs, is exclusively in the Harvey ras gene. Differential ras gene expression was examined in mammary tissue from virgin, pregnant, and lactating rats. Harvey ras expression was 1.5-2-fold higher than Kirsten ras or N-ras at each adult stage of development, with the highest ras levels expressed during pregnancy. The modest difference in total mRNA expression found between the independent members of the ras gene family is unlikely to fully account for the exclusive tissue-specificity of Harvey ras activation observed in rat mammary carcinogenesis. Thus, the role of Ras protein specificity was studied by infecting the mammary gland of virgin rats in situ with replication-defective retroviral vectors expressing either the activated or wild-type forms of Harvey- or Kirsten-ras. A 7-14-fold higher number of mammary carcinomas was observed after infection with vectors expressing the G35 to A activated Harvey ras gene product compared with those expressing G35 to A activated Kirsten ras. Mammary carcinomas also developed from infusion of vectors expressing wild-type Harvey ras, but not wild-type Kirsten ras. These data suggest the importance of the Ras protein itself in determining the specificity of the highly homologous Ras family members in organ-specific carcinogenesis.

Animals↗
PubMed1998

On the statistical analysis of allelic-loss data.

This paper concerns the statistical analysis of certain binary data arising in molecular studies of cancer. In allelic-loss experiments, tumour cell genomes are analysed at informative molecular marker loci to identify deleted chromosomal regions. The resulting binary data are used to infer properties of putative suppressor genes, genes involved in normal cell cycling. Various factors can complicate this inference, including background loss of heterozygosity, spatial (that is, within chromosome) dependence of the binary responses, non-informativeness of markers, covariates such as protein levels or tumour histology, heterogeneity of cells within tumours, and measurement error. We focus on the first three factors, discussing methods for statistical inference that separate background loss from significant loss. We outline the extension to other inferences, such as comparison questions and the relationship to covariates. Using characteristic features of tumourigenesis, we present a framework for the stochastic modelling of allelic-loss data, and build models within this framework; in particular, we propose a simple model that has chromosome breaks at locations of a Poisson process, and preferential selection cells with inactivated suppressor genes. We illustrate these methods on allelic-loss data from induced rat mammary tumours and human bladder cancers.

Animals↗
PubMed1998

Mapping of 55 new rat microsatellite markers from chromosome-specific libraries.

Fifty-five novel rat microsatellite markers were isolated from libraries specific for rat chromosomes (Chrs) 1, 2, and 7. The markers were mapped in three backcross rat populations. Thirty of these markers mapped to Chrs 1, 2, or 7, while the other 25 mapped to other chromosomes. New markers for two genes, liver-specific transporter gene (Livtr) and insulin-responsive glucose transporter (Glut4), were also mapped to rat Chrs 9 and 10, respectively. Three provisionally assigned markers from previous studies were also confirmed. Detailed methodologies for the generation and enrichment of clones containing repeat sequences and for the isolation of chromosome-specific markers are presented, since they represent unique combinations and modifications of previous protocols. Such methods and the newly presented markers should be useful for both specific and general mapping studies in the rat.

Animals↗
PubMed1998

Modulation of small G protein isoprenylation by anticancer monoterpenes in in situ mammary gland epithelial cells.

Monoterpenes have been shown to both prevent and treat mammary cancer in animal models and are currently in clinical testing in advanced cancer patients. In this study, we investigated a biochemical modulation associated with the antitumor activity of monoterpenes, the inhibition of protein isoprenylation in monoterpene chemoprevention target tissue, i.e. the in situ mammary gland epithelial cells. We first developed a new methodology that for the first time permitted the study of protein isoprenylation and other products in the mevalonate pathway in in situ mammary cells. Using this approach, we found that chronically feeding rats with an anticancer dose of perillyl alcohol resulted in a 22% inhibition of coenzyme Q synthesis and a 19% inhibition of small G protein isoprenylation in mammary gland epithelial cells in situ. The greatest inhibition of small G protein isoprenylation observed was the 28% inhibition of isoprenylation of RhoA by type I geranylgeranyl protein transferase (GGPTase). Given that some substrates of type I GGPTase, such as RhoA and Rac1, have transforming properties, the possibility that the inhibition of type I GGPTase will change the cellular location and functionality of these proteins and thus contribute to the chemoprevention activity of monoterpenes, is discussed.

Animals↗
PubMed1998

Genetic identification of multiple loci that control breast cancer susceptibility in the rat.

We have used a rat model of induced mammary carcinomas in an effort to identify breast cancer susceptibility genes. Using genetic crosses between the carcinoma-resistant Copenhagen (COP) and carcinoma-sensitive Wistar-Furth rats, we have confirmed the identification of the Mcs1 locus that modulates tumor number. We have now also identified two additional loci, Mcs2 and Mcs3. These three loci map to chromosomes 2, 7, and 1, respectively, and interact additively to suppress mammary carcinoma development in the COP strain. They are responsible for a major portion of the tumor-resistant phenotype of the COP rat. No loss of heterozygosity was observed surrounding the three loci. A fourth COP locus, Mcs4, has also been identified on chromosome 8 and acts in contrast to increase the number of carcinomas. These results show that mammary carcinoma susceptibility in the COP rat is a polygenic trait. Interestingly, a polymorphism in the human genomic region homologous to the rat Mcs4 region is associated with an increased breast cancer risk in African-American women. The isolation of the Mcs genes may help elucidate novel mechanisms of carcinogenesis, provide information important for human breast cancer risk estimation, and also provide unique drug discovery targets for breast cancer prevention.

Alleles↗
PubMed1998

A double mirror W/C multilayer monochromator for radiation biology applications.

A double-mirror multilayer monochromator was developed for the purpose of irradiating live cell cultures at the Synchrotron Radiation Center, University of Wisconsin-Madison. The monochromator is designed for the soft x-ray region with photon energies between 270 and 2400 eV. Multilayer mirrors with 55 bilayers of W/C and a bilayer spacing of d = 3.0 nm are sputter deposited on Si substrates. By proper masking of the sputtering sources, variation in the bilayer spacing over the area of the mirror is minimized. The uniformity of the bilayer spacing was measured to be delta d/d < 1%, over the 75 mm x 25 mm area of the mirrors. The reflectivity was measured as a function of energy to determine the integrated reflectivity and evaluate the contribution of the specular reflection and higher orders to the monochromatic beam. The use of suitable filters with a Si(Li) detector allows determination of the spectral output of the monochromator. The output power of the monochromator between 270 and 2400 eV is measured. The resolution of the monochromator is delta lambda/lambda = 0.04. Applications of the monochromator to radiation biology are discussed.

Cells, Cultured↗
PubMed1998

Quantitative studies of ductal versus alveolar differentiation from rat mammary clonogens.

In a program aimed at defining and characterizing the cellular origins of radiogenic mammary cancer, we developed and have used a quantitative rat mammary cell transplantation model to investigate hormonal control of differentiation, growth, and response to ionizing radiation. In this model, in response to appropriate hormonal stimulation, a subpopulation of transplanted mammary epithelial cells gives rise to either alveolar colonies (AU) or ductal colonies (DU). The cumulative data support the conclusion that both types of colonies are derived from single clonogenic mammary cells. In the current experiments the glucocorticoid, cortisol, which does not induce mammary proliferation or differentiation alone, promoted AU formation with milk secretion from grafted clonogens when present with estrogen and/or pituitary MtH (primarily prolactin and growth hormone) from co-grafted MtT. Progesterone synergized with estrogen in a dose-dependent fashion in induction of DU formation in mammary cell grafts in ovariectomized MtT-co-grafted rats and antagonized glucocorticoid-dependent AU development in such grafts in adrenalectomized-ovariectomized MtT-co-grafted rats as well. We conclude that hormonal regulation of growth and differentiation of rat mammary glands in situ is mediated to a significant extent through effects on the mammary clonogens and their immediate progeny. The mammary clonogen transplantation model permits quantitative investigation of such hormone actions on a mammary clonogen basis in vivo.

Animals↗
PubMed1998

Phase I clinical trial of perillyl alcohol administered daily.

Perillyl alcohol (POH; NSC-641066), a naturally occurring monoterpene, has shown antitumor and preventive activity in preclinical studies in rodent models. Drug-related activities that have been observed include the induction of apoptosis, cell cycle arrest, the inhibition of posttranslational modification of proteins that are involved in signal transduction, and differential gene regulation. We treated 18 patients who had advanced malignancies with POH, which was given on a continuous three-times-a-day schedule at the following doses: (a) level 1 (L1), 800 mg/m2/dose; (b) level 2 (L2), 1600 mg/m2/dose; and (c) level 3 (L3), 2400 mg/m2/dose. The main toxicity, which seemed to be dose related, was gastrointestinal and included nausea and vomiting, anorexia, unpleasant taste, satiety, and eructation. Two heavily pretreated ovarian cancer patients experienced reversible > or =grade 3 granulocytopenia. Grade 1-2 fatigue was also noted. The parent drug was not detectable in the plasma. The mean peak plasma levels of the two main metabolites on days 1 and 29 were 175 and 139 microM (L1), 472 and 311 microM (L2), and 456 and 257 microM (L3) for perillic acid (PA) and 7.1 and 9.8 microM (L1), 34.2 and 34.0 microM (L2), and 26.2 and 23.4 microM (L3) for dihydroperillic acid (DHPA). Peak levels were noted 2-3 h postingestion for PA and 3-5 h postingestion for DHPA. Metabolite half-lives measured about 2 h for each. POH, PA, and DHPA were detectable in the urine of all patients at L3. About 9% of the total dose was recovered in the first 24 h. The majority was recovered as PA; less than 1% was recovered as POH. Disease stabilization for > or =6 months was seen, although no objective tumor responses were noted. Further study of POH continues with a more frequent dosing schedule.

Adult↗
PubMed1998

Synchroton-produced ultrasoft X rays: equivalent cell survival at the isoattenuating energies 273 eV and 860 eV.

In this paper we report on survival of Chinese hamster V79 and mouse C3H 10T1/2 cells after irradiation with synchrotron-produced 273 eV and 860 eV ultrasoft X rays. These two energies, which are available by multilayer monochromatization of the synchrotron output spectrum, exhibit equal attenuation within living cells. Such an isoattenuating energy pair allows the direct examination of how biological effectiveness varies with the energy of the ultrasoft X rays. In comparing survival results, we find similar biological effectiveness of these two energies for both the C3H 10T1/2 and the V79 cells. These results are not consistent with previous findings of increasing RBE with decreasing ultrasoft X-ray energies. In addition, after correcting for mean nuclear dose based on measurements of cell thickness obtained with confocal microscopy, we find no significant differences in survival between the two ultrasoft X-ray energies and 250 kVp X rays. These results suggest that RBE does not increase with decreasing energy of ultrasoft X rays between 860 eV and 273 eV. The possible impact of our results on past results for ultrasoft X rays is discussed.

Animals↗
PubMed1998

A comparison of cytotoxicity after whole- or partial-cell irradiation with synchrotron-produced ultrasoft X rays.

We introduce a method which allows partial-volume irradiation of live cells using synchrotron-produced ultrasoft X rays and micro-fabricated irradiation masks. The masks were made by X-ray lithography at the University of Wisconsin Synchrotron Radiation Center, and they consist of 1.85-microm-wide stripes of gold 1.35 microm apart plated onto thin silicon nitride membranes. When placed adjacent to Mylar on which live cells are plated, these masks allow cells to be irradiated in a striped pattern with dimensions much smaller than the cell nuclei. Using 1340 eV synchrotron-produced X rays, we compare the survival of cells subjected to uniform irradiation and cells subjected to partial-volume irradiation. Our results show that, at equal mean dose to the nucleus (i.e. equal total energies deposited), survival is not statistically different for the two treatments over a wide range of doses. Thus imparting equal energies to smaller intranuclear volumes does not appear to enhance cell killing.

Animals↗
PubMed1997

Inhibition of type I and type II geranylgeranyl-protein transferases by the monoterpene perillyl alcohol in NIH3T3 cells.

The monoterpene perillyl alcohol has anticancer activities that include both prevention and treatment of a wide variety of cancers in animal models. In purified enzyme studies, perillyl alcohol inhibited farnesyl-protein transferase and type I geranylgeranyl-protein transferase. However, whether and which of the polyprenyl-protein transferases is inhibited by perillyl alcohol in vivo is not known. The previously reported monoterpene-induced inhibition of the incorporation of [14C]mevalonolactone into proteins in cultured cells could be due to an inhibition of one or several enzymes in the mevalonate pathway or to changes in the levels of protein substrates for isoprenylation. In the current study, we first analyzed the levels of individual phosphorylated isoprenoid intermediates between mevalonate and geranylgeranyl pyrophosphate in NIH3T3 cells labeled for 4 hr with [14C]mevalonolactone and found that perillyl alcohol did not inhibit the synthesis of these intermediates. Next, proteins including Ras, RhoA, and Rab6 were immunoprecipitated from NIH3T3 cells. Perillyl alcohol was found to inhibit the incorporation of [14C]mevalonolactone into RhoA and Rab6 but not Ras protein. The cellular levels of these three proteins were constant over the 4-hr treatment period. Finally, the distribution of Ras, Rap1, and Rab6 proteins between the aqueous and the detergent-enriched phases was measured. Rap1 and Rab6 but not Ras from perillyl alcohol-treated NIH3T3 cells accumulated in the aqueous phase. Thus, we conclude that perillyl alcohol can inhibit the in vivo prenylation of specific proteins by type I and type II geranylgeranyl-protein transferases but not farnesyl-protein transferase in NIH3T3 cells.

3T3 Cells↗
PubMed1997

The genetic penetrance of the activated neu oncogene for the induction of mammary cancer in vivo.

Carcinogenesis is most often viewed as a multistage disease process. An exception to this was suggested for neu transformation of mammary cells in a transgenic model (Muller et al., 1988); however, this interpretation is controversial (Bouchard et al., 1989). In order to better define neu mammary transformation in vivo, we directly measured the genetic penetrance of the neu oncogene. Mammary cells in situ were infected with replication-defective retroviral vectors carrying the activated neu oncogene (pJRneu). A limiting dilution in vivo transplantation assay was used to measure the percentage of mammary clonogenic (stem-like) cells that stably and functionally integrated this vector. Based on this, the percentage of clonogens integrating and expressing neu that could progress to mammary carcinomas was quantified to estimate the penetrance of this gene in mammary carcinogenesis. The genetic penetrance of neu was 3.6% (95% confidence interval 2.2%-5.8%). This high degree of genetic penetrance is compatible with the observations that certain neu-transgenic mice develop a very great number of mammary carcinomas (Muller et al., 1988). However, whether these data are compatible with a single-step transformation model (100% penetrance) is uncertain and is discussed.

Animals↗
PubMed1997

Cancer chemoprevention and therapy by monoterpenes.

Monoterpenes are found in the essential oils of many plants including fruits, vegetables, and herbs. They prevent the carcinogenesis process at both the initiation and promotion/progression stages. In addition, monoterpenes are effective in treating early and advanced cancers. Monoterpenes such as limonene and perillyl alcohol have been shown to prevent mammary, liver, lung, and'other cancers. These compounds have also been used to treat a variety of rodent cancers, including breast and pancreatic carcinomas. In addition, in vitro data suggest that they may be effective in treating neuroblastomas and leukemias. Both limonene and perillyl alcohol are currently being evaluated in phase I clinical trials in advanced cancer patients. The monoterpenes have several cellular and molecular activities that could potentially underlie their positive therapeutic index. The monoterpenes inhibit the isoprenylation of small G proteins. Such inhibitions could alter signal transduction and result in altered gene expression. The results of a new gene expression screen-subtractive display-have identified or confirmed several up- or downregulated genes in regressing mammary carcinomas. For example, these regressing tumors overexpress the mannose 6-phosphate/IGF II receptor. The product of the gene both degrades the mammary tumor mitogen IGF II and activates the cytostatic factor TGF-beta. These and other alterations in the gene expression of mammary carcinomas lead to a G1 cell cycle block, followed by apoptosis, redifferentiation, and finally complete tumor regression in which tumor parenchyma is replaced by stromal elements. It is likely that monoterpenes prevent mammary cancer during their progression stage by mechanisms similar to those that occur during therapy. In contrast, prevention of mammary cancer by polycyclic hydrocarbons such as 7,12-dimethylbenz[a]anthracene occur by the induction of detoxifying phase II hepatic enzymes.

Anticarcinogenic Agents↗
PubMed1996

Identifying differential gene expression in monoterpene-treated mammary carcinomas using subtractive display.

Monoterpene-induced/repressed genes were identified in regressing rat mammary carcinomas treated with dietary limonene using a newly developed method termed subtractive display. The subtractive display screen identified 42 monoterpene-induced genes comprising 9 known genes and 33 unidentified genes, as well as 58 monoterpene-repressed genes comprising 1 known gene and 57 unidentified genes. Several of the identified differentially expressed genes are involved in the mitoinhibitory transforming growth factor beta signal tranduction pathway, as demonstrated by isolation of the mannose 6-phosphate/insulin-like growth factor II receptor and the transforming growth factor beta type II receptor. The monoterpene-induced/repressed genes indicate that apoptosis and differentiation act in concert to effect carcinoma regression. Apoptosis is suggested by the cloning of a marker of programmed cell death, lipocortin 1. Consistent with a differentiation/remodeling process occurring during tumor regression, subtractive display identified YWK-II and neuroligin 1. Thus far, of the cDNAs putatively identified as differentially expressed in this complex in situ carcinoma model, 5 were tested, and each one has been confirmed to be differentially expressed. Additionally, many of the identified known genes are expressed as rare transcripts and exhibit small but significant changes in abundance. Together, these points demonstrate the unique utility of this new gene expression screen to identify altered gene expression in a complex in vivo environment.

Animals↗
PubMed1996

Allelic imbalance in mammary carcinomas induced by either 7,12-dimethylbenz[a]anthracene or ionizing radiation in rats carrying genes conferring differential susceptibilities to mammary carcinogenesis.

To identify and compare the genetic lesions associated with tumorigenesis in rats carrying the mammary carcinoma suppressor (MCS) 1 gene, we induced mammary carcinomas in (Wistar Furth (WF) x Copenhagen (Cop))F1 rats by using either 7,12-dimethylbenz[a]anthracene (DMBA) or radiation. The tumors were screened for allelic imbalances by using polymerase chain reaction and 65 polymorphic microsatellite markers spanning the genome. No allelic imbalance was detected at the mapped location of MCS-1 on chromosome 2; however, a scan of the genome revealed random allelic imbalances in the radiation-induced tumors. In addition, non-random loss of heterozygosity (LOH) on chromosome 1 in the DMBA-induced tumors was documented. We then screened three other subsets of DMBA- and radiation-induced mammary carcinomas from (WF x Fischer (F344))F1, (Wistar Kyoto x F344)F1, and (F344 x Cop)F1 rats for imbalance on chromosomes 1 and 2. No allelic imbalance was detected in the MCS-1 region of chromosome 2 in any of the tumors screened. Nonrandom imbalance on chromosome 1 was detected but only in the DMBA-induced tumors from the (F344 x Cop)F1 rats. Thus, only Cop-derived F1 rats have mammary tumors with the chromosome 1 imbalance; however, the imbalance does not favor the Cop parental allele. We also analyzed the DMBA-induced tumors with LOH at chromosome 1 for Ha-ras codon 61 mutation and found no association. These results suggest that loss of the MCS-1 Cop allele is not required for tumor formation, that the genetic background of the F1 rat appears to influence the type of genetic lesion identified in the mammary tumors, and that there is no association between Ha-ras codon 61 mutation and chromosome 1 imbalance in our model system.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1996

Cloning, genetic mapping and expression studies of the rat Brca1 gene.

The breast cancer gene BRCA1 has previously been cloned from both human and mouse. We cloned a fragment of the rat Brca1 homologue in order to map it and explore its biological function. Partial cDNA fragments of the rat Brca1 homologue were isolated by RT-PCR. Sequence analysis revealed that the RING-finger domain is well conserved among rat, mouse and human. Rat Brca1 mRNA was expressed in most tissues studied with the highest level in testis, consistent with studies in human and mouse. Next, intron 6-containing DNA fragments were amplified by PCR from WKY and WF rat strains. The splicing sites between exon 6 and exon 7 are conserved between rat and human. Partial sequencing of the rat Brca1 intron 6 revealed a polymorphism of a pentanucleotide TTTTG repeat between the WKY and WF strains. With this intragenic microsatellite marker, we were able to map precisely the rat Brca1 gene to chromosome 10 using a genetic linkage study of (WKY x WF)F1 x WF backcross rats. Brca1 cosegregates with marker BAND3A, and is flanked by R5123 and R5842. Using this polymorphic marker, we also investigated the loss of heterozygosity (LOH) of the Brca1 microsatellite marker in carcinogen- or radiation-induced mammary carcinomas in (WF x F344)F1 female rats. No LOH or somatic microsatellite instability was detected in 18 DMBA-induced tumors studied. Only one LOH of the F344 allele was observed in 26 radiation-induced tumors tested. Ribonuclease protection assays demonstrated that Brca1 mRNA levels are similar in normal rat mammary glands and mammary carcinomas of various etiologies, including those induced by DMBA, NMU, activated-neu and activated-ras oncogenes.

Alleles↗