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Biomedical subjects

R Clarke

Publications and source records attributed to R Clarke.

At least 73 records · Page 4Linked to original sources
PubMed1999

Differential distribution of protein phosphatase 2A in human breast carcinoma cell lines and its relation to estrogen receptor status.

Protein phosphatase 2A (PP2A) acts as a growth suppressor and is negatively influenced by oncogenic signals. We determined its activity in various human breast carcinoma (HBC) cell types to understand its relationship to estrogen receptor (ER) expression as well as to the distribution of protein kinase C (PKC), an opposing enzyme. PP2A activity was measured using a preferred substrate, histone H1 phosphorylated by PKC. PP2A activity was higher in both the soluble and nuclear fractions of ER-positive cell lines (MCF-7, T47D and ZR-75-1) than in the ER-negative cell lines (MDA-MB-231, Hs578T and BT-20). PP2A multiple forms (2A0, 2A1, 2A2), separated by DEAE-cellulose chromatography and immunoblot analysis of PP2A catalytic subunit, also showed similar differences in these two HBC cell types. In all cases, PP2A distribution was inversely correlated with the PKC activity profile. Moreover, PP2A activity in MCF-7 cells maintained in estrogen-depleted medium was low. Nonetheless, it was induced by a prolonged treatment with 17beta-estradiol, this induction being blocked by the antiestrogens, tamoxifen and ICI-182,780. Studies in both MCF-7 transfectants stably overexpressing ras and MDA-MB-231 transfectants stably expressing ER, suggested that a low PP2A distribution in ER-negative HBC cell types may be related to tumor progression rather than the loss of ER. Conceivably, the presence of high PP2A along with low PKC in ER-positive HBC cell types may be related to the restricted cell growth associated with the retention of a certain degree of differentiation or hormonal control. Conversely, the presence of low PP2A along with high PKC in ER-negative cell types may be related to hormone-independent enhanced cell growth.

Breast Neoplasms↗
PubMed1999

Reversal of tamoxifen resistance of human breast carcinomas in vivo by neutralizing antibodies to transforming growth factor-beta.

BACKGROUND: Overexpression of transforming growth factor (TGF)-beta has been reported in human breast carcinomas resistant to antiestrogen tamoxifen, but the role of TGF-beta in this resistant phenotype is unclear. We investigated whether inhibition of TGF-beta2, which is overexpressed in LCC2 tamoxifen-resistant human breast cancer cells, could modify antiestrogen resistance. METHODS: TGF-beta2 expression was evaluated in LCC2 cells and tamoxifen-sensitive LCC1 cells by northern blot analysis. Secreted TGF-beta activity was quantified by use of an 125I-TGF-beta competitive radioreceptor assay. Sensitivity to tamoxifen was measured in a soft agarose colony-forming assay and in a xenograft model in nude and beige/nude mice. Natural killer (NK) cell cytotoxicity was measured by 51Cr release from LCC1 and LCC2 cell targets coincubated with human peripheral blood mononuclear cells. Decrease in TGF-beta2 expression in LCC2 cells was achieved by treatment with antisense oligodeoxynucleotides and confirmed by TGF-beta2 immunoblot analysis. RESULTS AND CONCLUSIONS: The proliferative response of LCC2 cells to tamoxifen in vitro was not altered by TGF-beta neutralizing antibodies. However, established LCC2 tumors in nude mice treated with tamoxifen plus TGF-beta antibodies failed to grow, whereas tumors treated with tamoxifen plus a control antibody continued to proliferate. This reversal of tamoxifen resistance by TGF-beta antibodies did not occur in beige/nude mice, which lack NK-cell function, suggesting that immune mechanisms may be involved in the antitumor effects of tamoxifen. Antisense TGF-beta2 oligodeoxynucleotides enhanced the NK sensitivity of LCC2 cells in the presence of tamoxifen. Finally, LCC1 tumors were markedly more sensitive to tamoxifen in NK-active than in NK-deficient mice. IMPLICATIONS: These data suggest that host NK function mediates, in part, the antitumor effect of tamoxifen and that TGF-beta2 may abrogate this mechanism, thus contributing to tamoxifen resistance.

Animals↗
PubMed1999

Growth and phenotypic characterization of porcine coronary artery smooth muscle cells.

Vascular smooth muscle cell (VSMC) proliferation significantly contributes to atherosclerotic plaque formation and limits the success rate of percutaneous transluminal coronary angioplasty. We derived a population of porcine coronary artery SMCs to characterize VSMC proliferation and phenotype in preparation to study the molecular actions of VSMC mitogens and antiproliferative agents. Growth assays were designed to minimize the estrogen content in the culture medium, since this steroid hormone significantly influences VSMC growth and the expression of VSMC mitogens and their receptors. Culture conditions were identified such that this criterion was achieved while maintaining a significant VSMC growth rate. Cells cultured in serum-free medium, regardless of growth factor supplements, did not remain adherent to a plastic culture substrate, nor did they proliferate. Dextran-coated charcoal (DCC)-treated sera, including fetal bovine, calf, and porcine, supported VSMC adhesion, but not growth. Whole fetal bovine serum (FBS) produced the best proliferative response. A type-I collagen-coated culture surface significantly enhanced VSMC growth, but only in culture medium containing non-DCC-treated FBS. Flow cytometry analyses confirmed the mitogenic effects of this substrate. The VSMCs exhibited a morphological change on type-I collagen, but this was not accompanied by a change in VSMC phenotype. Our data indicate that culture of these porcine coronary artery SMCs in 2.5% FBS plus 10 ng platelet-derived growth factor-BB per ml in phenol red-free medium on type-I collagen may be the optimal conditions for studying the molecular aspects of VSMC mitogens and antiproliferative agents.

Animals↗
PubMed1999

Inhibition of estrogen receptor function promotes porcine coronary artery smooth muscle cell proliferation.

Estradiol-17beta (E2) can inhibit vascular smooth muscle cell (VSMC) proliferation probably through its ability to activate its nuclear estrogen receptors (ER). Activation or inhibition of the ER by cognate permissive or non-permissive ligands, respectively, would indicate whether ER action is critical for this vascular protective effect. We investigated a previously characterized population of cultured porcine coronary artery SMCs for ER expression and for the response of these cells to estrogens and antiestrogens. Reverse transcription-polymerase chain reaction and Western blot analyses demonstrated ER mRNA and protein, respectively, in these cells. While the culture conditions required may have prevented the demonstration of physiological effects of E2, the antiestrogens, ICI 182,780 and 4-hydroxytamoxifen, stimulated VSMC proliferation. The data suggest that, by interrupting ER function, antiestrogens significantly increased the VSMC mitotic rate. This model may be used to identify ER-regulated genes that function to control the growth of these coronary artery SMCs.

Animals↗
PubMed1999

Septic arthritis caused by Corynebacterium amycolatum following vascular graft sepsis.

A case of septic arthritis caused by Corynebacterium amycolatum in a native hip joint occurred in an adult man following contralateral vascular graft sepsis, and was successfully treated with intravenous vancomycin followed by oral doxycycline and rifampicin. To the authors' knowledge, this is the only reported case of septic arthritis due to C. amycolatum.

Anti-Bacterial Agents↗
PubMed1999

The influence of maternal diet on breast cancer risk among female offspring.

The induction of breast cancer is a long process, containing a series of biological events that drive a normal mammary cell towards malignant growth. However, it is not known when the initiation of breast cancer occurs. One hypothesis is that a high estrogenic environment during the perinatal period increases subsequent breast cancer risk. There are many sources of extragonadal estrogens, particularly in the diet. The purpose of this paper is to review the evidence that a high maternal intake of dietary fats increases serum estrogens during pregnancy and increases breast cancer risk in daughters. Our animal studies show that a high maternal consumption of corn oil consisting mainly of linoleic acid (omega-6 polyunsaturated fatty acid, PUFA), increases both circulating estradiol (E2) levels during pregnancy and the risk of developing carcinogen-induced mammary tumors among the female rat offspring. A similar increase in breast cancer risk occurs in female offspring exposed to injections of E2 through their pregnant mother. Our data suggest that the mechanisms by which an early exposure to dietary fat and/or estrogens increases breast cancer risk is related to reduced differentiation of the mammary epithelial tree and increased number of mammary epithelial cell structures that are known to the sites of neoplastic transformation. These findings may reflect our data of the reduced estrogen receptor protein levels and protein kinase C activity in the developing mammary glands of female rats exposed to a high-fat diet in utero. In summary, a high dietary linoleic acid intake can elevate pregnancy estrogen levels and this, possibly by altering mammary gland morphology and expression of fat- and/or estrogen-regulated genes, can increase breast cancer risk in the offspring. If true for women, breast cancer prevention in daughters may include modulating the mother's pregnancy intake of some dietary fats.

Animals↗
PubMed1999

Inhibition of P-glycoprotein activity and reversal of multidrug resistance in vitro by rosemary extract.

The transmembrane transport pump P-glycoprotein (Pgp) causes the efflux of chemotherapeutic agents from cells and is believed to be an important mechanism in multidrug resistance (MDR) in mammary tumours. In the present study we demonstrate that an extract of the common dietary herb rosemary (Rosemarinus officinalis Labiatae), increases the intracellular accumulation of commonly used chemotherapeutic agents, including doxorubicin (DOX) and vinblastine (VIN), in drug-resistant MCF-7 human breast cancer cells which express Pgp. Rosemary extract (RE) inhibits the efflux of DOX and VIN, which are known to be substrates of Pgp, but does not affect accumulation or efflux of DOX in wild type MCF-7 cells, which lack Pgp. Treatment of drug-resistant cells with RE increases their sensitivity to DOX, which is consistent with an increased intracellular accumulation of the drug. RE blocks the binding of the VIN analogue azidopine to Pgp. Thus, it appears that RE directly inhibits Pgp activity by inhibiting the binding of drugs to Pgp.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1999

LCC15-MB: a vimentin-positive human breast cancer cell line from a femoral bone metastasis.

The LCC15-MB cell line was established from a femoral bone metastasis that arose in a 29-year-old woman initially diagnosed with an infiltrating ductal mammary adenocarcinoma. The tumor had a relatively high (8%) S-phase fraction and 1/23 positive lymph nodes (LN). Both the primary tumor and LN metastasis were positive for estrogen receptor (ER) and progesterone receptor (PgR), but lacked erbB2 expression. Approximately one year later, the patient presented with a 0.8 cm comedo-type intraductal mammary adenocarcinoma in the left breast that was negative for ER and PgR, but positive for erbB2. Thirty-five months after the initial diagnosis she was treated for acute skeletal metastasis, and stabilized with a hip replacement. At this time, tumor cells were removed from surplus involved bone, inoculated into cell culture, and developed into the LCC 15-MB cell line. The bone metastasis was a poorly differentiated adenocarcinoma lacking ER, PgR, and erbB2, characteristics shared by the LCC15-MB cells, although ER can be re-expressed by treatment of the LCC15-MB cells for 5 days with 75 microM 5-aza-2'-deoxycytidine. The LCC15-MB cell line is tumorigenic when implanted subcutaneously in NCr nu/nu mice and produces long-bone metastases after intracardiac injection. Although the bone metastasis from which the LCC15-MB cell line was derived lacked vimentin (VIM) expression, the original primary tumor and lymph node metastasis were strongly VIM positive, as are LCC15-MB cells in vitro and in nude mice. The karyotype and isozyme profiles of LCC15-MB cells are consistent with its origin from a human female, with most chromosome counts in the hypertriploid range. Thirty-two marker chromosomes are present. These cells provide an in vitro/in vivo model in which to study the inter-relationships between ER, VIM, and bone metastasis in human breast cancer.

Adult↗
PubMed1999

Prepubertal exposure to zearalenone or genistein reduces mammary tumorigenesis.

Prepubertal exposure to a pharmacological dose (500 mg kg(-1)) of the phyto-oestrogen genistein can reduce the incidence and multiplicity of carcinogen-induced mammary tumours in rats. However, such an exposure also disrupts the function of the hypothalamic-pituitary-gonadal axis, making it unsuitable for breast cancer prevention. We studied whether prepubertal exposure to genistein at a total body dose broadly comparable to the level typical of Oriental countries, approximately 1 mg kg(-1) body weight, affects mammary tumorigenesis. We also studied whether prepubertal exposure to zearalenone, a major source for phyto-oestrogens in the USA, influences breast cancer risk. Prepubertal rats were treated between postnatal days 7 and 20, with 20 microg (approximately 1 mg kg(-1) body weight) of either genistein or zearalenone. Zearalenone exposure significantly reduced both the incidence and multiplicity of mammary tumours induced by 7,12-dimethylbenz(a)anthracene (DMBA). Genistein exposure significantly reduced tumour multiplicity, but not tumour incidence, when compared with vehicle-treated animals. Furthermore, 60% of the tumours in the genistein group were not malignant, while all the tumours analysed for histopathology in the vehicle and zearalenone groups were adenocarcinomas. A higher number of differentiated alveolar buds, and lower number of terminal ducts, were present in the DMBA-treated mammary glands of the phyto-oestrogen exposed rats. The concentration of oestrogen receptor (ER) binding sites after the DMBA treatment was low in the mammary glands of all groups but a significantly higher proportion of the glands in the zearalenone exposed rats were ER-positive (i.e. ER levels > or = 5 fmol mg(-1) protein) than the glands of the vehicle controls. Our data suggest that a prepubertal exposure to a low dose of either zearalenone or genistein may protect the mammary gland from carcinogen-induced malignant transformation, possibly by increasing differentiation of the mammary epithelial tree.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1999

Control of low-level radiation exposure: time for a change?

The carcinogenic risks of exposure to low-level ionising radiation used by the ICRP have been challenged as being, at the same time, both too high and too low. This paper explains that the epidemiological evidence will always be limited at low doses, so that understanding the cellular mechanisms of carcinogenesis is increasingly important to assess the biological risks. An analysis is then given of the reasons why the challenges to the ICRP, especially about the linear non-threshold response model, have arisen. As a result of considering the issues, the Main Commission of the ICRP is now proposing a revised, simpler, approach based on the concept of what is being called 'controllable dose'. This is an individual-based philosophy and represents a shift in emphasis by the Commission from societal-oriented criteria using Collective Dose. Finally the paper speculates on the consequences for radiological protection of such a change in policy. The Commission wishes its ideas to be discussed as part of its reconsideration of its recommendations.

Dose-Response Relationship, Radiation↗
PubMed1999

Naturally occurring extracellular matrix as a scaffold for musculoskeletal repair.

The use of naturally occurring extracellular matrix materials as scaffolds for the repair and regeneration of tissues is receiving increased attention. The present study evaluates the use of the extracellular matrix derived from porcine small intestinal submucosa as a scaffold for anterior cruciate ligament replacement in a goat model. Sixty healthy adult female goats were divided into two equal groups of 30 each. The right anterior cruciate ligament of each goat was removed surgically and replaced with either a patellar tendon autograft or a small intestinal submucosa anterior cruciate ligament scaffold. Three animals from each group were sacrificed at 6 weeks, 3 months, 6 months, and 1 year after surgery and grafts were harvested for histopathologic examination. Six animals from each group were sacrificed immediately after surgery, 3 months, and 1 year after surgery and the grafts were harvested for biomechanical testing. There was no evidence for an adverse clinical response to the xenogeneic small intestinal submucosa scaffold. Anterior drawer values were not different between the two groups at any point. The failure force of the patellar tendon autograft increased from 253 N at Time 0 to 879 N at 12 months. The failure force for the small intestinal submucosa repair device was 721 N at Time 0, decreased to 293 N at 3 months, followed by an increase to 706 N at 12 months. Histopathologic analysis showed a mixed inflammatory cell presence within the small intestinal submucosa scaffold including macrophages and lymphocytes in the early months after surgery. The inflammatory cells disappeared in the later stages of remodeling and the histologic appearance of the small intestinal submucosa remodeled grafts and the patellar tendon autografts were indistinguishable at 12 months. Xenogeneic small intestinal submucosa holds promise as a resorbable bioscaffold for anterior cruciate ligament repair in the goat model.

Animals↗
PubMed1998

The LCC15-MB human breast cancer cell line expresses osteopontin and exhibits an invasive and metastatic phenotype.

We have characterized the LCC15-MB cell line which was recently derived from a breast carcinoma metastasis resected from the femur of a 29-year-old woman. LCC15-MB cells are vimentin (VIM) positive, exhibit a stellate morphology in routine cell culture, and form penetrating colonies when embedded in three-dimensional gels of Matrigel or fibrillar collagen. They show high levels of activity in the Boyden chamber chemomigration and chemoinvasion assays, and like other invasive human breast cancer (HBC) cell lines, LCC15-MB cells activate matrix-metalloproteinase-2 in response to treatment with concanavalin A. In addition, these cells are tumorigenic when implanted subcutaneously in nude mice and recolonize bone after arterial injection. Interestingly, both the primary lesion and the bone metastasis from which LCC15-MB were derived, as well as the resultant cell line, abundantly express the bone matrix protein osteopontin (OPN). OPN is also expressed by the highly metastatic MDA-MB-435 cells, but not other invasive or noninvasive HBC cell lines. Expression of OPN is retained in the subcutaneous xenograft and intraosseous metastases of LCC15-MB as detected by immunohistochemistry. Both VIM and OPN expression have been associated with breast cancer invasion and metastasis, and their expression by the LCC15-MB cell line is consistent with its derivation from a highly aggressive breast cancer. These cells provide a useful model for studying molecular mechanisms important for breast cancer metastasis to bone and, in particular, the implication(s) of OPN and VIM expression in this process.

Adult↗
PubMed1998

Folate, vitamin B12, and serum total homocysteine levels in confirmed Alzheimer disease.

BACKGROUND: Recent studies suggest that vascular disease may contribute to the cause of Alzheimer disease (AD). Since elevated plasma total homocysteine (tHcy) level is a risk factor for vascular disease, it may also be relevant to AD. OBJECTIVE: To examine the association of AD with blood levels of tHcy, and its biological determinants folate and vitamin B12. DESIGN: Case-control study of 164 patients, aged 55 years or older, with a clinical diagnosis of dementia of Alzheimer type (DAT), including 76 patients with histologically confirmed AD and 108 control subjects. SETTING: Referral population to a hospital clinic between July 1988 and April 1996. MAIN OUTCOME MEASURES: Serum tHcy, folate, and vitamin B12 levels in patients and controls at entry; the odds ratio of DAT or confirmed AD with elevated tHcy or low vitamin levels; and the rate of disease progression in relation to tHcy levels at entry. RESULTS: Serum tHcy levels were significantly higher and serum folate and vitamin B12 levels were lower in patients with DAT and patients with histologically confirmed AD than in controls. The odds ratio of confirmed AD associated with a tHcy level in the top third (> or = 14 micromol/L) compared with the bottom third (< or = 11 micromol/L) of the control distribution was 4.5 (95% confidence interval, 2.2-9.2), after adjustment for age, sex, social class, cigarette smoking, and apolipoprotein E epsilon4. The corresponding odds ratio for the lower third compared with the upper third of serum folate distribution was 3.3 (95% confidence interval, 1.8-6.3) and of vitamin B12 distribution was 4.3 (95% confidence interval, 2.1-8.8). The mean tHcy levels were unaltered by duration of symptoms before enrollment and were stable for several years afterward. In a 3-year follow-up of patients with DAT, radiological evidence of disease progression was greater among those with higher tHcy levels at entry. CONCLUSIONS: Low blood levels of folate and vitamin B12, and elevated tHcy levels were associated with AD. The stability of tHcy levels over time and lack of relationship with duration of symptoms argue against these findings being a consequence of disease and warrant further studies to assess the clinical relevance of these associations for AD.

Aged↗