Recent advances in the molecular genetics of hereditary breast and ovarian cancer.
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Breast Neoplasms↗
Biomedical subjects
R W Wiseman
Publications and source records attributed to R W Wiseman.
Brca1 is expressed independently of hormonal stimulation in the mouse ovary.
BRCA1 mutations lead to cancer susceptibility in hormonally dependent tissues such as the ovary and breast. To test the hypothesis that Brca1 expression in the ovary is hormonally regulated and specifically regulated by a functional estrogen receptor, we examined its expression by in situ hybridization in ovaries from virgin, pregnant, and lactating mice, in hypophysectomized mice treated with hormones, and in estrogen-receptor-deficient mice. To determine the relationship between Brca1 expression and cell cycle, serial and adjacent sections of ovary were evaluated for proliferating cell nuclear antigen by immunohistochemistry. Regardless of the model, Brca1 was consistently expressed in granulosa and thecal cells of follicle populations that proliferate independently of hormonal stimulation. Expression was similar in these same follicle populations in the ovaries of estrogen-receptor-deficient mice, in which the lack of this estrogen receptor results in abnormal and incomplete follicular development. Brca1 expression was diminished in the granulosa and thecal cells of hormonally dependent antral follicles. Brca1 expression was also localized to luteal cells of recently formed corpora lutea and corpora lutea associated with pregnancy, but it was greatly diminished in regressing corpora lutea in cycling mice. In all cases, Brca1 expression correlated to S-phase proliferating cell nuclear antigen nuclear staining. Thus, Brca1 expression in the mouse ovary occurs independently of hormonal status and in the absence of a major estrogen receptor-mediated pathway; it is, however, closely correlated with cell cycle in mouse ovarian granulosa, thecal, and luteal cell.
Animals↗
Dideoxy fingerprinting assay for BRCA1 mutation analysis.
Since the isolation of BRCA1, the familial breast/ovarian cancer predisposition gene, much effort has been invested in characterizing the mutation spectrum. The large size of the gene and the wide distribution of its more than 100 mutations has increased the challenge of this endeavor such that traditional mutation detection techniques are inadequate. We examined the sensitivity of dideoxy fingerprinting (DDF), which combine a Sanger sequencing reaction with multiple-fragment single-strand conformation analysis (SSCA), as a mutation detection technique to screen BRCA1. Here we describe the technique and compare its sensitivity with that of SSCA in detecting 21 previously described BRCA1 sequence variants. All the variants were detected by DDF, but only 17 of 21 (81%) were observed by SSCA under standard conditions. Three of four alterations missed by SSCA were base substitutions. As a BRCA1 mutation detection technique, DDF was more sensitive than SSCA and may prove to be a useful research tool in defining the mutation spectrum within this and other genes.
Breast Neoplasms↗
Non-invasive quantitative 31P MRS assay of mitochondrial function in skeletal muscle in situ.
A method for non-invasive, quantitative 31P NMR spectroscopic investigation of mitochondrial function in human skeletal muscle in situ is described. High time resolution 31P NMR measurements of phosphocreatine, inorganic phosphate and ATP resonances were conducted on human forearm flexor muscle during involuntary twitch contraction at eight different frequencies. Mitochondrial and glyco(geno)lytic ATP synthesis fluxes, and the cytosolic free energy of ATP hydrolysis (delta GP), were calculated at incremental steady-states of energy balance. The covariation of mitochondrial ATP synthesis flux, JPMOP, and delta GP was quasi-linear over the physiological range of free energy values. Curve-fit analysis of the covariation yielded a maximal sustainable JPMOP of 0.24 +/- 0.06 mmol ATP l-1.s-1 and a midpoint potential, (delta GP)0.5, of 58.1 +/- 1.2 kJ/mole in the muscle cells.
Adult↗
Phosphorus metabolite distribution in skeletal muscle: quantitative bioenergetics using creatine analogs.
The functional coupling of contractile activity to metabolic processes in skeletal, cardiac and smooth muscle has been extensively examined in the intact cell through the advent of 31P-NMR spectroscopy. The near-equilibrium formulation for creatine kinase (CK) has been used for the calculation of ADPfree and ATP chemical potential in many of these studies. However, control of the bulk cytoplasmic PCr/Cr ratio by the ATP/ADP ratio through CK implies that the ATP/ADP ratio is the same in all loci within the cell. Alternatively the cytoplasmic fraction of ATP and ADP must be so large that other 'compartments' do not influence the physicochemical properties of the bulk cytoplasm. By feeding creatine analogs to rodents, it is possible to test whether these synthetic analogs and the endogenous substrates obey simple rules of enzyme kinetics and equilibration. Two important concepts can be tested: (1) Are phosphorus metabolises fully visible to in vivo 31P-NMR measurements? (2) Does CK equilibrate with its substrates in the cytosol? It will be shown that in spite of localized enzymatic activity and microcompartments in the cell (such as mitochondria), creatine kinase equilibrates with its substrates in fast and slow skeletal muscles at rest. Therefore, the physicochemical properties of the cytosol are best described as freely mixing with respect to the bioenergetically important metabolites (PCr, ATP and phospho-analog) and fully quantifiable by 31P-NMR spectroscopy. It follows that only a narrow range of intercellular heterogeneity with respect to chemical potential is acceptable if bioenergetic processes (e.g. CK fluxes or regulation of oxidative phosphorylation) are to be meaningfully interpreted in a rigorous biochemical framework.
Animals↗
Skeletal myoblast transplantation for repair of myocardial necrosis.
Myocardial infarcts heal by scarring because myocardium cannot regenerate. To determine if skeletal myoblasts could establish new contractile tissue, hearts of adult inbred rats were injured by freeze-thaw, and 3-4.5 x 10(6) neonatal skeletal muscle cells were transplanted immediately thereafter. At 1 d the graft cells were proliferating and did not express myosin heavy chain (MHC). By 3 d, multinucleated myotubes were present which expressed both embryonic and fast fiber MHCs. At 2 wk, electron microscopy demonstrated possible satellite stem cells. By 7 wk the grafts began expressing beta-MHC, a hallmark of the slow fiber phenotype; coexpression of embryonic, fast, and beta-MHC continued through 3 mo. Transplanting myoblasts 1 wk after injury yielded comparable results, except that grafts expressed beta-MHC sooner (by 2 wk). Grafts never expressed cardiac-specific MHC-alpha. Wounds containing 2-wk-old myoblast grafts contracted when stimulated ex vivo, and high frequency stimulation induced tetanus. Furthermore, the grafts could perform a cardiac-like duty cycle, alternating tetanus and relaxation, for at least 6 min. Thus, skeletal myoblasts can establish new muscle tissue when grafted into injured hearts, and this muscle can contract when stimulated electrically. Because the grafts convert to fatigue-resistant, slow twitch fibers, this new muscle may be suited to a cardiac work load.
Animals↗
The signal transduction function for oxidative phosphorylation is at least second order in ADP.
To maintain ATP constant in the cell, mitochondria must sense cellular ATP utilization and transduce this demand to F0-F1-ATPase. In spite of a considerable research effort over the past three decades, no combination of signal(s) and kinetic function has emerged with the power to explain ATP homeostasis in all mammalian cells. We studied this signal transduction problem in intact human muscle using 31P NMR spectroscopy. We find that the apparent kinetic order of the transduction function of the signal cytosolic ADP concentration ([ADP]) is at least second order and not first order as has been assumed. We show that amplified mitochondrial sensitivity to cytosolic [ADP] harmonizes with in vitro kinetics of [ADP] stimulation of respiration and explains ATP homeostasis also in mouse liver and canine heart. This result may well be generalizable to all mammalian cells.
Adenosine Diphosphate↗
Homozygous codeletion and differential decreased expression of p15INK4b, p16INK4a-alpha and p16INK4a-beta in mouse lung tumor cells.
The genes of murine cyclin D-dependent kinase inhibitors, p15INK4b and p16INK4a, are located in a region of chromosome 4 where overlapping deletions were found in lung adenocarcinomas. The p16INK4a gene uniquely consists of alternative first exons (E1alpha and E1beta), which are spliced to exon 2 in alternative reading frames to either encode p16INK4a (alpha form) or another potential tumor suppressor, p19ARF (beta form). We examined 99 lung adenocarcinomas of C3H/HeJ x A/J F1(C3AF1) and A/J x C3H/HeJ F1(AC3F1) mouse hybrids and 18 (13 metastatic, 5 nonmetastatic) tumorigenic mouse lung epithelial cell lines for p15INK4b and p16INK4a gene inactivation. Homozygous codeletion occurred in eight of the 13 (62%) metastatic, four of the five (80%) nonmetastatic cell lines, but in only six of 99 (6%) adenocarcinomas. Neither p15INK4b nor p16INK4a gene was individually deleted in any of the tumors or cell lines, and all deletions of the p16INK4a gene extended into exon 2, which would be expected to disrupt the functions of both p16INK4a and p19ARF. We also detected no intragenic mutations of either gene in 44 tumors that displayed loss of heterozygosity at the p16INK4a locus or in any of the cell lines. Transcript levels of p16INK4a-alpha, p16INK4a-beta and p15INK4b also were examined in each of the cell lines that retained copies of these genes. Whereas an immortal mouse lung epithelial cell line (E10) and two metastatic tumor cell lines (LM1 and E9) expressed p16INK4a-beta and p15INK4b mRNA, the alpha transcript of p16INK4a was detected in only the LM1 cell line. These results suggest that both p15INK4b and p16INK4a (alpha and beta) are targets of inactivation in mouse lung tumorigenesis.
Adenocarcinoma↗
Allelotype analysis of 2',3'-dideoxycytidine- and 1,3-butadiene-induced lymphomas in B6C3F1 mice.
To identify potential tumor suppressor genes involved in lymphoma development, we generated allelotypes of 16 2',3'-dideoxycytidine (ddC and 31 1,3-butadiene (BD)-induced lymphomas from C57BL/6 x C3H/He F1 (hereafter called B6C3F1) mice. Two or more anonymous simple sequence length polymorphisms per autosome were examined for loss of heterozygosity (LOH). Allelic losses throughout the genome were generally infrequent, except for markers on chromosome 2, 4, 11 and 12. The highest frequency of allelic losses was observed on chromosome 12, with 38 and 39% in ddC and BD-induced lymphomas, respectively. The most prevalent LOH was localized to the distal region bounded by markers D12Mit263 and D12Nds2. No known tumor suppressor genes have been mapped to this region, and no obvious candidates could be identified, suggesting the presence of novel suppressor gene(s). LOH on chromosome 2 was observed in 31% of ddC-induced lymphomas but in only 3% (1/31) of BD-induced lymphomas, suggesting a ddC-specific genetic effect. Detailed analysis localized a potential tumor suppressor gene residing on the distal region of chromosome 2, between markers D2Mit147 and D2Mit148. Twenty-five % of ddC-induced and 23% of BD-induced lymphomas showed LOH on chromosome 4, and two discrete regions were identified. One of the regions includes the IFN gene cluster and is syntenic to human chromosome 9p2l-22. Candidate tumor suppressor genes, Mts1 (multiple tumor suppressor 1) and Mts2 have been mapped to this region. The second region is located on the distal part of chromosome 4, which is homologous to human chromosome 1p35-36, a region that is frequently deleted in various types of human tumors. Finally, 19% of ddC-induced and 29% of BD-induced lymphomas revealed LOH on chromosome 11 at the Acrb locus, which lies within 1 cM of p53, suggesting that the p53 tumor suppressor gene also plays a role in lymphomagenesis. These results suggest that multiple potential suppressor loci contribute to lymphoma development in B6C3F1 mice.
Alleles↗
Detection of BRCA1 mutations in women with early-onset ovarian cancer by use of the protein truncation test.
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BRCA1 Protein↗
Microsomal epoxide hydrolase polymorphism as a risk factor for ovarian cancer.
Microsomal epoxide hydrolase (EPHX) is one of many enzymes involved in the metabolism of endogenous and exogenous toxicants. Polymorphic forms of the human EPHX gene have been described that vary in enzymatic activity, and one, Tyr113His, has been associated with hepatocellular carcinoma susceptibility. We demonstrated that EPHX was highly expressed in the human ovary, and investigated whether specific EPHX genotypes are associated with ovarian cancer susceptibility. Seventy-three Caucasian patients with ovarian cancer and 75 Caucasian-female controls without cancer were genotyped for the Tyr113His polymorphism by a polymerase chain reaction-restriction fragment length polymorphism assay. The frequency of the homozygous high-activity genotype was 41% in the control population and 64% in the ovarian cancer patients. The odds ratio for ovarian cancer with this genotype was 2.6 (95% confidence interval 1.3, 5.0; P < 0.01). The increased ovarian cancer risk associated with the high-activity genotype could reflect differences in metabolic activation of endogenous or exogenous carcinogens.
Blotting, Northern↗
Preservation of phosphagen kinase function during transient hypoxia via enzyme abundance or resistance to oxidative inactivation.
Mammalian lactate dehydrogenase and phosphofructokinase are more susceptible in vitro to superoxide (O2) and hydroxyl (.OH) radicals than pyruvate kinase and glucose-6-phosphate dehydrogenase, suggesting that differential inactivation of regulatory enzymes contributes to the metabolic disintegration in stenoxic tissues during transient hypoxia. Likewise, creatine kinase in smooth muscle from porcine ileum is significantly reduced by hypoxia-reoxygenation ex vivo from 300 (+/- 18.2 SE, n = 8) to 196 U.g wet wt-1 (+/- 16.7, P < 0.001, ANOVA). Conversely, arginine kinase, from the myocardium of Limulus polyphemus, a species that tolerates anoxia for days was 2.9-fold less susceptible to oxidative inactivation. To examine whether preservation of kinase function is related to euryoxic capacity, a combination of non-invasive 31P-NMR spectroscopy and enzyme-linked assays was used to follow ATP and phosphagen status during hypoxia-reoxygenation in porcine ileum smooth muscle, L. polyphemus myocardium, and the myocardium of Argopecten irradians, a scallop species tolerant of hypoxia for only 24 h. Despite wide differences in phylogeny, euryoxic capacity and oxidative vulnerability of the phosphagen kinases, in all three tissues, the phosphagen pool recovered concomitant with ATP during reoxygenation, thereby revealing competent kinase function. In the mammalian tissue, such preservation of kinase function is facilitated by a 2400-fold excess of enzyme activity.
Animals↗
An inevitable dilemma: prenatal testing for mutations in the BRCA1 breast-ovarian cancer susceptibility gene.
The BRCA1 breast-ovarian cancer susceptibility gene was identified recently. Germline mutations in BRCA1 may be responsible for as many as 5% of breast and ovarian cancers. Inherited alterations confer up to a 94% risk of developing breast and/or ovarian cancer by age 70. With the discovery of BRCA1, there will be a heavy demand for genetic testing. Because of the large size of the gene and the distribution of reported mutations, scientists face considerable technical problems in developing widely available screening tests; clinicians will face even greater ethical problems in applying them. In the context of research programs, women with BRCA1 mutations are already being identified, and their physicians are confronted with a number of complex medical, ethical, legal, and social issues. Obstetricians will be faced with counseling parents regarding prenatal testing for specific BRCA1 mutations. Although it is difficult to formulate straightforward guidelines regarding prenatal BRCA1 testing, clinicians and health care providers must be familiar with the nuances of the debate so that these issues can be discussed wisely with patients. As with many ethically challenging problems in medicine, individual clinicians and their patients will have to work together to determine the course of action with which they are most comfortable. Although elective termination of a pregnancy with a germline mutation in BRCA1 is an option, experience with other adult-onset diseases suggests that only a minority of parents will choose this option.
BRCA1 Protein↗
BRCA2 mutations in primary breast and ovarian cancers.
The second hereditary breast cancer gene, BRCA2, was recently isolated. Germline mutations of this gene predispose carriers to breast cancer, and, to a lesser extent, ovarian cancer. Loss of heterozygosity (LOH) at the BRCA2 locus has been observed in 30-40% of sporadic breast and ovarian tumours, implying that BRCA2 may act as a tumour suppressor gene in a proportion of sporadic cases. To define the role of BRCA2 in sporadic breast and ovarian cancer, we screened the entire gene for mutations using a combination of techniques in 70 primary breast carcinomas and in 55 primary epithelial ovarian carcinomas. Our analysis revealed alterations in 2/70 breast tumours and none of the ovarian carcinomas. One alteration found in the breast cancers was a 2-basepair (bp) deletion (4710delAG) which was subsequently shown to be a germline mutation, the other was a somatic missense mutation (Asp3095Glu) of unknown significance. Our results suggest that BRCA2 is a very infrequent target for somatic inactivation in breast and ovarian carcinomas, similar to the results obtained for BRCA1.
Aged↗
Role of BRCA1 mutation screening in the management of familial ovarian cancer.
Families with multiple cases of ovarian cancer have long been observed, and in the past prophylactic oophorectomy has been advocated for women with a history of ovarian cancer in two first-degree relatives. It is now thought that > 90% of familial ovarian cancer is due to inherited mutations in the BRCA1 breast-ovarian cancer susceptibility gene on chromosome 17q. BRCA1 testing is being performed in several academic medical centers on a research basis and is also now commercially available. With the ability to identify inherited mutations in BRCA1, prophylactic oophorectomy and other interventions intended to decrease cancer mortality can be offered specifically to women who carry a mutation, but the optimal strategy for decreasing cancer mortality in BRCA1 families has not yet been determined. To facilitate further clinical and basic research in this field, our group and others have established multidisciplinary hereditary breast-ovarian cancer clinics that offer a wide range of services including BRCA1 testing, genetic counseling, and cancer prevention and treatment.
Chromosomes, Human, Pair 17↗
Effect of intracellular pH on force development depends on temperature in intact skeletal muscle from mouse.
The cellular mechanism of muscle fatigue is still in debate. Opposite conclusions regarding the role of intracellular pH (pHi) in fatigue have been drawn from skinned fiber vs. isolated perfused muscle studies. Because these experiments are typically performed at different temperatures, we tested the hypothesis that temperature alters the effects of pH on force. Tetanic force of isolated mouse extensor digitorum longus was measured at temperatures between 13 and 25 degrees C in either normocapnia (5% CO2) or hypercapnia (25% CO2). Hypercapnia decreased pHi (monitored by 31P nuclear magnetic resonance spectroscopy) by the same amount at both 15 and 25 degrees C. However, inhibition of force by hypercapnia was greater at the lower temperature. A similar pattern of temperature-dependent inhibition of force by pH was observed in glycerinated fibers from rabbit psoas at maximum Ca2+ activation. We conclude that temperature differences are responsible for disparate conclusions on the role of pHi in muscle fatigue. Based on our results, we suggest that changes in pHi may have little or no role in the loss in force production associated with muscular fatigue at physiological temperatures.
Animals↗
BRCA1 expression is induced before DNA synthesis in both normal and tumor-derived breast cells.
Insight into the function of the BRCA1 tumor suppressor gene may be gained by studying its regulation. In this study, the expression of BRCA1 was examined as a function of the cell cycle in normal and tumor-derived breast epithelial cells. Cells arrested in G(zero) or early in G1 contained low levels of BRCA1 mRNA. After release, populations of cells reached maximal levels of BRCA1 in late G1 and S phase. Induction of BRCA1 was shown to occur before the onset of DNA synthesis by synchronizing cells at the G1-S boundary. Levels of the BRCA1 protein were regulated in a similar manner. No difference was observed between primary cultures of normal mammary epithelial cells and immortalized tumor-derived cell lines. These results suggest that BRCA1 may function at the G1-S checkpoint.
Adult↗
Regulation of microfilament organization and anchorage-independent growth by tropomyosin 1.
Variants of chemically immortalized Syrian hamster embryo cells that had either retained (supB+) or lost (supB-) the ability to suppress tumorigenicity when hybridized with a fibrosarcoma cell line were subcloned. Both supB cell types are nontumorigenic; however, the supB- but not supB+ cells exhibit conditional anchorage-independent growth. Alterations of actin microfilament organization were observed in supB- but not supB+ cells that corresponded to a significant reduction of the actin-binding protein tropomyosin 1 (TM-1) in subB- cells. To examine the possibility of a direct relationship between TM-1 expression and the subB- phenotype, subB+ cells were transfected with an expression vector containing the TM-1 cDNA in an antisense orientation. The antisense-induced reduction of TM-1 levels in supB+ clones caused a microfilament reorganization and conferred anchorage-independent growth potential that were indistinguishable from those characteristic of supB- cells. These data provide direct evidence that TM-1 regulates both microfilament organization and anchorage-independent growth and suggest that microfilament alterations are sufficient for anchorage-independent growth.
Actin Cytoskeleton↗