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Biomedical subjects

R L Sutherland

Publications and source records attributed to R L Sutherland.

At least 91 records · Page 5Linked to original sources
PubMed1995

Expression and regulation of cyclin genes in breast cancer.

Cyclins, the regulatory subunits of cyclin-dependent kinases, control passage through key check-points within the cell cycle. Since dysregulated expression and function of cyclins can lead to loss of normal growth control some of these genes are oncogenes. We have studied cyclin gene expression, regulation and function in breast cancers. Induction of cyclin D1 is an early event in mitogenic stimulation of breast cancer cells by growth factors and steroids. Furthermore, inhibition of cyclin D1 expression is an early response to growth inhibition by antioestrogens. Ectopic expression of cyclin D1 in T-47D breast cancer cells demonstrated that cyclin D1 is rate-limiting for progression through G1 phase and is sufficient for growth arrested cells to complete the cell cycle. Since this gene is frequently overexpressed in human breast cancers it may contribute to the development and progression of some breast carcinomas.

Breast Neoplasms↗
PubMed1995

Heregulin (HRG)-induced mitogenic signaling and cytotoxic activity of a HRG/PE40 ligand toxin in human breast cancer cells.

The heregulins (HRGs) are a family of growth factors that bind direction to erbB3 and erbB4 and induce tyrosine phosphorylation of erbB2 via receptor heterodimerization. Since erbB2, erbB3, and erbB4 (erbB2-4) are often overexpressed in human breast cancer cells, we produced recombinant HRGs and a HRG-based ligand toxin to investigate the signaling events triggered by HRGs and the ability of these ligands to specifically target such cells. Recombinant HRG beta 2 stimulated the tyrosine phosphorylation of erbB2-4 in ZR-75-1 human breast cancer cells. This was accompanied by the tyrosine phosphorylation of Shc and the formation of complexes between Shc and the adapter protein Grb2. Complexes were also detected between Shc and erbB2-4. However, GRb2 was detected in erbB2 and erbB4 but not erbB3 immunoprecipitates. Thus, these receptors exhibit mechanistic differences in their coupling to Ras signaling, and HRG beta 2 administration triggers multiple inputs into the Ras signaling pathway, involving receptor-Grb2, receptor-Shc, and Shc-Grb2 complexes. HRG beta 2 addition also stimulated the association of erbB3 with phosphatidylinositol-3-kinase. In accordance with the activation of key mitogenic signaling pathways, HRG beta 2 stimulated the proliferation of MCF-7 and T-47D human breast cancer cells. Moreover, when tested for the ability to stimulate cell cycle re-entry of T-47D cells arrested under serum-free conditions, HRG beta 2 was more effective than insulin, previously the most potent mitogen identified using this system. Finally, a HRG beta 2 PE40 ligand toxin was constructed and found to exhibit cytotoxic activity against human breast cancer cells overexpressing erbB3 alone or in combination with erbB4 and/or erbB2.

ADP Ribose Transferases↗
PubMed1994

Cyclin D1 induction in breast cancer cells shortens G1 and is sufficient for cells arrested in G1 to complete the cell cycle.

The sequential transcriptional activation of cyclins, the regulatory subunits of cell-cycle-specific kinases, is thought to regulate progress through the cell cycle. Cyclins are therefore potential oncogenes, and cyclin D1 overexpression and/or amplification at its genomic locus, 11q13, are common features of several human cancers. Induction of cyclin D1 is an early response to mitogenic stimulation in several cell types, but the consequences of altered expression of this gene in human cells of epithelial origin remain undefined. We assessed the effects of alterations of cyclin D1 expression in human breast cancer cells by generating T-47D cells expressing human cyclin D1 under the control of a zinc-responsive metallothionein promoter. In cycling cells induction of cyclin D1 after zinc treatment resulted in an increase in the number of cells progressing through G1 and in the rate of transition from G1 to S phase, indicating that cyclin D1 is rate-limiting for progress through G1 phase. In cells arrested in early G1 phase after growth factor deprivation, zinc induction of cyclin D1 was sufficient for completion of the cell cycle, a process requiring growth factor stimulation in control cells. These data demonstrate a critical role for cyclin D1 in human breast cancer cell-cycle control and suggest that deregulated expression of cyclin D1 is likely to reduce dependence on normal physiological growth stimuli, thereby providing a growth advantage to tumor cells and a potential mechanism of resistance to endocrine therapy.

Blotting, Northern↗
PubMed1994

Antiestrogen regulation of cell cycle progression and cyclin D1 gene expression in MCF-7 human breast cancer cells.

The molecular mechanisms by which antiestrogens inhibit breast cancer cell proliferation are not well understood. Using cultured breast cancer cell lines, we studied the effects of antiestrogens on proliferation and cell cycle progression and used this information to select candidate cell cycle regulatory genes that are potential targets for antiestrogens. Under estrogen- and serum-free conditions antiestrogens inhibited proliferation of MCF-7 cells stimulated with insulin. Cells were blocked at a point in G1 phase. These effects are comparable with those in serum- and estrogen-containing medium and were also seen to a lesser degree in nude mice bearing MCF-7 tumors. Similar observations with other peptide mitogens suggest that the process inhibited by antiestrogens is common to estrogen and growth factor activated pathways. Other studies have identified G1 cyclins as potential targets for growth factor and steroid hormone/steroid antagonist regulation of breast epithelial cell proliferation. In MCF-7 cells growing in the presence of fetal calf serum, cyclin D1 mRNA was rapidly down-regulated by steroidal and nonsteroidal antiestrogens by an apparently estrogen receptor mediated mechanism. Cyclin D1 gene expression was maximally inhibited before effects on entry into S phase and inhibition was therefore not merely a consequence of changes in cell cycle progression. Together with data on the effects of antiestrogens in serum-free conditions [1], these results suggest down-regulation of cyclin D1 by antiestrogens may be a general phenomenon in estrogen receptor-positive breast cancer cells, independent of culture conditions and class of antiestrogen. These observations are compatible with the hypothesis that reductions in cyclin D1 levels may mediate in part the action of antiestrogens in blocking entry of cells into S phase.

Animals↗
PubMed1994

MDA-MB-453, an androgen-responsive human breast carcinoma cell line with high level androgen receptor expression.

The role of androgens and the androgen receptor (AR) in the development and progression of breast cancer is poorly understood. To further define a potential model for androgen action in breast cancer, MDA-MB-453 cells, which express AR in the absence of oestrogen receptors and progesterone receptors, were further characterised in terms of AR expression and androgen responsiveness. High level expression of AR was confirmed by northern blot analysis, radioligand binding and immunocytochemistry, and could not be accounted for by AR gene amplification. Three endogenous androgen-responsive genes (fatty acid synthetase, gross cystic disease fluid protein of 15 kDa and prolactin receptor) and a transfected reporter gene, containing an androgen-responsive element, were induced following androgen administration. A synthetic androgen, mibolerone, induced moderate (27% above control) stimulation of MDA-MB-453 cell proliferation, which was abrogated by the simultaneous administration of the synthetic androgen antagonist, anandron, demonstrating that the effect was AR-mediated. In summary, MDA-MB-453 cells express high levels of functional AR, and thus provide a valuable in vitro model for further studies on androgen regulation of gene expression, and perhaps cell proliferation in breast cancer.

Blotting, Southern↗
PubMed1994

Cell cycle control by steroid hormones.

Steroid hormones have well documented stimulatory and inhibitory effects on target cell proliferation. These effects are steroid- and target cell-specific and are mediated by cell cycle phase-specific actions. The molecular mechanisms by which steroids control rates of cell cycle progression are, however, not well defined. Recent advances in our understanding of cell cycle control by proto-oncogenes, tumor suppressor genes and cyclin dependent kinases provide models for further delineating the molecular basis of proliferation control by steroids. Transcriptional activation of c-fos, c-myc and cyclin D1 by estrogens and progestins in breast cancer cells and inhibition of expression of these genes by antiestrogens provide a paradigm for further understanding cell cycle control by steroids.

Animals↗
PubMed1994

Activation of the Ras signalling pathway in human breast cancer cells overexpressing erbB-2.

The c-erbB-2 proto-oncogene encodes a receptor tyrosine kinase (RTK) closely related to the epidermal growth factor receptor (EGFR). Overexpression of erbB-2 occurs in approximately 20% of human breast tumours, where increased expression correlates with poor patient prognosis. The EGFR is coupled to the Ras signalling pathway by interaction with the adaptor protein Grb2, and Sos, a Ras GDP-GTP exchange factor. In this study, activation of the erbB-2 receptor and its association with Grb2 and Sos was investigated in breast cancer cell lines which overexpress erbB-2. The receptor was found to be tyrosine phosphorylated in all cell lines in which it is overexpressed. Western blotting of Grb2 and Sos immuneprecipitates from such cells revealed co-precipitation of erbB-2, demonstrating association of the Grb2/Sos complex with erbB-2 in vivo. Furthermore, a fusion protein containing only the SH2 domain of Grb2 bound to erbB-2 immobilized on nitrocellulose, indicating that association with Grb2 is direct and mediated by the SH2 domain of Grb2. The degree of association between the erbB-2 receptor and Grb2 in vivo was related to erbB-2 overexpression, and MAP kinase, which functions downstream from Ras, displayed markedly increased activity in cell lines overexpressing erbB-2. These results demonstrate that erbB-2 is coupled to Ras signalling via the Grb2/Sos complex, and that overexpression of this receptor in breast cancer cells leads to amplification of the Ras signalling pathway.

Adaptor Proteins, Signal Transducing↗
PubMed1994

Overexpression of the Grb2 gene in human breast cancer cell lines.

A receptor blotting technique was used to detect SH2 domain containing epidermal growth factor receptor (EGFR) substrates that exhibited differential expression either between normal breast epithelial cells and breast cancer cells or between different human breast cancer cell lines. This identified a 25 kD protein, subsequently identified as Grb2, which was markedly overexpressed in three breast cancer cell lines (MCF-7, MDA-MB-361 and -453) relative to both normal breast epithelial cells and the majority of breast cancer cell lines. Northern blot analysis revealed that 7/19 breast cancer cell lines exhibited more than twofold overexpression of Grb2 mRNA, with overexpression correlating with high expression of erbB receptors. In MCF-7, MDA-MB-361 and -453 cells the overexpression of Grb2 mRNA and protein was accompanied by a small amplification of the Grb2 gene locus. Overexpression of Grb2 correlated with increased complex formation between Grb2 and the hSos-1 Ras GDP-GTP exchange protein. This upregulation of the Ras signalling pathway might modulate the growth factor sensitivity of human breast cancer cells and therefore play a role in tumour progression.

Adaptor Proteins, Signal Transducing↗
PubMed1993

Estradiol induction of retinoic acid receptors in human breast cancer cells.

Retinoic acid inhibits proliferation and steroid receptor gene expression in human breast cancer cell lines. Retinoic acid receptors (RAR)alpha, -beta, and -gamma are expressed in these cells and the expression of RAR alpha is significantly greater in estrogen receptor (ER)-positive cells. This study was undertaken to determine whether the same relationship between RAR alpha and ER gene expression was present in human breast cancers and to explore the possibility that the higher level of RAR alpha in ER-positive cells was due to estrogen regulation of RAR alpha gene expression. RAR alpha and ER mRNA expression were determined by Northern blot analysis in 116 primary breast tumors; 94 (81%) tumors were ER-positive and of these 87 (93%) were also RAR alpha-positive. The coexpression of ER and RAR alpha was statistically significant (P = 0.0052 by chi 2 contingency analysis). There was also a positive correlation (by linear regression analysis) between the levels of expression of ER and RAR alpha mRNA (r2 = 0.251, P = 0.0001), which confirmed the relationship previously documented in breast cancer cell lines and suggested that RAR alpha expression may be modulated in breast cancer in vivo by estrogens acting via the ER. The ability of estradiol to regulate RAR alpha gene expression was examined in vitro using T-47D cells which had been rendered sensitive to estrogen by repeated passage in steroid-depleted medium. Estradiol increased RAR alpha gene expression, but not that of RAR beta or RAR gamma, in a concentration-dependent manner, with the effect being maximal at 10(-10) M and less marked at higher concentrations. The effect was rapid, being detectable 1 h after and maximal 6 h after treatment with 10(-10) M estradiol. Co-treatment of cells with estradiol and antiestrogens (tamoxifen or ICI 164384, 4 x 10(-7) M for 6 h) inhibited the estradiol induction of RAR alpha gene expression, demonstrating that the effect was ER mediated. The estradiol sensitivity of the effect was underscored by the demonstration that addition of untreated serum to cells growing under steroid-depleted conditions was sufficient to induce maximal RAR alpha gene expression. This effect was totally abolished by addition of ICI 164384. In summary, the demonstration that estradiol increased RAR alpha mRNA levels in breast cancer cells supports the hypothesis that the correlation between RAR alpha and ER gene expression in breast tumors and breast cancer cell lines is due to estradiol augmentation of RAR alpha gene expression.

Breast Neoplasms↗
PubMed1993

Effects of the progestin antagonist RU 486 on T-47D breast cancer cell cycle kinetics and cell cycle regulatory genes.

Possible mechanisms by which the progestin antagonist RU 486 inhibits cell growth were investigated by comparing the effects of the antiprogestin with those of progestin and antiestrogen. Exposure of T-47D breast cancer cells to RU 486 caused a decline in the proportion of cells in S phase, indicative of a block to cell cycle progression in G1 phase. This was accompanied by a marked decrease in c-myc expression but no change in cyclin D1 expression. The cell kinetic data suggest that progestin antagonist inhibition of proliferation and progestin stimulation of proliferation are mediated by opposing effects on the same mechanism. Both estrogen antagonists and progestin antagonists appear to act at a similar part of G1 phase but there are clear differences in their effects on cyclin D1 expression, suggesting that the mechanisms by which these compounds inhibit proliferation are distinct.

Breast Neoplasms↗
PubMed1993

Regulation of prolactin receptor expression by the tumour promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate in human breast cancer cells.

In both the normal and malignant human breast, cellular sensitivity to the proliferative and differentiative activities of the lactogenic hormones is conferred by expression of the prolactin receptor (PRLR). The PRLR is regulated by steroid hormones; however, recent findings have suggested that PRLR may also be regulated by protein kinase C. To examine this possibility we have studied the effect of various modulators of PKC activity on PRLR binding activity and gene expression in five PRLR positive human breast cancer cell lines. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), a tumour promoter and modulator of PKC activity, decreased PRLR binding activity in all cell lines examined. In MCF-7 cells, 10 nM TPA caused a 70% loss of PRLR mRNA after 12 h, paralleled 3 h later by a comparable loss of cell surface PRLR. Mezerein, a non-phorbol ester modulator of PKC activity and 1,2-dioctanoyl-sn-glycerol, a permeant analogue of the endogenous activator of PKC, also reduced PRLR binding activity and gene expression in a time- and concentration-dependent manner. Cycloheximide failed to abrogate the TPA-induced decline in PRLR mRNA levels, indicating that this process was not dependent upon continuing protein synthesis. No change in the stability of PRLR mRNA was observed during 24 h of TPA treatment and TPA reduced the rate of PRLR gene transcription within 3 h of treatment. These results demonstrate that modulators of PKC activity reduce PRLR binding activity and gene expression, implicating this signal transduction pathway in PRLR regulation.

Breast Neoplasms↗
PubMed1993

Acute effects of growth factors on T-47D breast cancer cell cycle progression.

Growth factors play a major role in the control of human breast cancer cell proliferation but their acute effects on cell cycle progression have not been well studied in these cells. T-47D cells, growth-inhibited by serum deprivation, were induced to re-enter the cell cycle in a concentration- and time-dependent manner by addition of insulin, insulin-like growth factor (IGF)-I, epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha) or basic fibroblast growth factor (bFGF). After a lag of approximately 10 h semi-synchronous entry into S phase was observed. The relative potencies of maximal concentrations of growth factors were in the order: insulin approximately IGF-I approximately bFGF > TGF alpha > EGF, identifying bFGF as among the most potent mitogens for these cells. Insulin or IGF-I alone resulted in growth rates comparable with those observed in fetal calf serum. These data demonstrate that single growth factors can induce a significant proportion of T-47D cells to traverse the cell cycle. The kinetics for entry into S phase were similar, indicating that the basis of differential sensitivity to the growth factors tested was the proportion of cells that responded and ultimately entered S phase.

Breast Neoplasms↗
PubMed1993

Cyclin gene expression and growth control in normal and neoplastic human breast epithelium.

Recent advances in defining the molecular mechanisms of cell cycle control in eukaryotes provide a basis for better understanding the hormonal control of cell proliferation in normal and neoplastic breast epithelium. It is now clear that a number of critical steps in cell cycle progression are controlled by families of serine/threonine kinases, the cdks. These kinases are activated by interactions with various cyclin gene products which form the regulatory subunits of the kinase complexes. Several families of cyclins control cell cycle progression in G1 phase, cyclins C, D and E, or in S, G2 and mitosis, cyclins A and B. Recent studies have defined the expression and regulation of cyclin genes in normal breast epithelial cells and in breast cancer cell lines. Following growth arrest of T-47D breast cancer cells by serum deprivation restimulation with insulin results in sequential induction of cyclin genes. Cyclin D1 mRNA increases within 1 h of mitogenic stimulation and is followed by increased expression of cyclins D3 and E in G1 phase, cyclin A in late G1/early S phase and cyclin B1 in G2. Similar results were observed following epidermal growth factor stimulation of normal breast epithelial cells. Other hormones--oestrogens and progestins--and growth factors--insulin-like growth factor-I and basic fibroblast growth factor--with actions in G1 were also investigated for their effects on G1 cyclin gene expression. In all cases there was an excellent correlation between the induction of cyclin D1 mRNA and subsequent entry into S phase. Furthermore, growth inhibition by antioestrogens and concurrent G1 arrest were preceded by an acute decrease in cyclin D1 gene expression. These observations suggest a likely role for cyclin D1 in mediating many of the known hormonal effects on cell proliferation in breast epithelial cells.

Breast↗
PubMed1993

Growth factor, steroid, and steroid antagonist regulation of cyclin gene expression associated with changes in T-47D human breast cancer cell cycle progression.

Cyclins and proto-oncogenes including c-myc have been implicated in eukaryotic cell cycle control. The role of cyclins in steroidal regulation of cell proliferation is unknown, but a role for c-myc has been suggested. This study investigated the relationship between regulation of T-47D breast cancer cell cycle progression, particularly by steroids and their antagonists, and changes in the levels of expression of these genes. Sequential induction of cyclins D1 (early G1 phase), D3, E, A (late G1-early S phase), and B1 (G2 phase) was observed following insulin stimulation of cell cycle progression in serum-free medium. Transient acceleration of G1-phase cells by progestin was also accompanied by rapid induction of cyclin D1, apparent within 2 h. This early induction of cyclin D1 and the ability of delayed administration of antiprogestin to antagonize progestin-induced increases in both cyclin D1 mRNA and the proportion of cells in S phase support a central role for cyclin D1 in mediating the mitogenic response in T-47D cells. Compatible with this hypothesis, antiestrogen treatment reduced the expression of cyclin D1 approximately 8 h before changes in cell cycle phase distribution accompanying growth inhibition. In the absence of progestin, antiprogestin treatment inhibited T-47D cell cycle progression but in contrast did not decrease cyclin D1 expression. Thus, changes in cyclin D1 gene expression are often, but not invariably, associated with changes in the rate of T-47D breast cancer cell cycle progression. However, both antiestrogen and antiprogestin depleted c-myc mRNA by > 80% within 2 h. These data suggest the involvement of both cyclin D1 and c-myc in the steroidal control of breast cancer cell cycle progression.

Breast Neoplasms↗
PubMed1993

Serum gamma-glutamyltransferase activity and protein concentration at birth and after suckling in calves with adequate and inadequate passive transfer of immunoglobulin G.

In an effort to characterize the activity of serum gamma-glutamyltransferase (GGT) in newborn calves before and after suckling and to explore the usefulness of serum GGT as an indicator of failure of passive transfer in calves, blood samples were collected from the first calves of 48 cows at the time of birth and at 1 day of age. Serum was harvested, and concentrations of IgG and protein and activity of GGT were determined. Morbidity and mortality events were monitored from birth to weaning. Calves suckling colostrum had 10 and 1.3 times greater serum concentrations of IgG and protein, respectively, and a 26 times greater serum activity of GGT, compared with concentrations at birth. Increases in GGT activity and protein concentration were correlated to increases in IgG concentration. Calves classified as having failure of passive transfer (< 800 mg of IgG/dl) had a 9.5 times greater risk of becoming sick prior to weaning, compared with calves determined to have partial failure of passive transfer and clinically normal calves (P = 0.0004). The sensitivity and specificity of a cutoff value of 200 IU of GGT/L of serum for diagnosing failure of passive transfer were 80 and 97%, respectively. The sensitivity and specificity of a cutoff value of 4.2 g of protein/dl serum for diagnosing failure of passive transfer were 80 and 100%, respectively. The Kappa values for diagnosis of failure of passive transfer, using serum concentrations of IgG vs activity of GGT, IgG vs protein, and GGT vs protein, were 0.72, 0.86, and 0.79, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Expression and amplification of cyclin genes in human breast cancer.

Cyclins, the regulatory subunits of cyclin-dependent kinases, play an important role in the control of cellular proliferation. Since dysregulated expression of these genes may contribute to the malignant phenotype the expression and amplification of cyclin A, B1, C, D1, D2, D3 and E genes were studied in 20 breast cancer cell lines. Increased expression of one or more of the cyclin A, B1, D1 or E genes was found in seven cell lines (35%); of these five (25%) showed increased expression of cyclin D1. Overexpression occurred in both the presence and absence of gene amplification. Conversely, amplification did not invariably lead to overexpression. Cyclin D2 expression was lower in breast cancer cell lines than in cultured normal breast epithelial cells. Cyclin D1 expression was further investigated in breast tumour biopsies: 56 of 124 specimens (45%) expressed higher levels of cyclin D1 mRNA than normal breast tissue. These data implicate dysregulated expression of several cyclin genes, particularly cyclin D1, as a potential factor in the pathogenesis of breast cancer.

Base Sequence↗
PubMed1993

Comparative effects of gold on the interactions of transcription factors with DNA.

We have previously hypothesized that a mode of action of the anti-rheumatic gold salt, aurothiomalate (AuTM), is the inhibition of DNA binding by transcription factors. Studies of the progesterone receptor (PR), which has a zinc finger structure in the DNA binding domain, were consistent with this hypothesis (1). Here we show that AuTM also markedly inhibits DNA binding by the transcription factor AP-1 and has less potent effects for AP-2, NF-1 and TFIID.

Cells, Cultured↗