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E A Musgrove

Publications and source records attributed to E A Musgrove.

At least 37 records · Page 2Linked to original sources
PubMed1997

Expression of the PEA3 group of ETS-related transcription factors in human breast-cancer cells.

The PEA3 group of transcription factors belongs to the ets family and is composed of 3 known members, PEA3, ERM and ER81, which are more than 95% identical within the DNA-binding ETS domain and exhibit 50% aa identity overall. Recently, transgenic mice bearing the c-erbB-2/neu oncogene have been shown to over-express PEA3 mRNA in mammary adenocarcinomas, suggesting a role for this gene family in mammary tumorigenesis. In the present work we characterized the mRNA expression levels of PEA3-group genes in a series of human epithelial breast cell lines. Each of the 3 genes was highly expressed in normal human HMEC 1001-7 and HMEC 219-4 cells. In breast-cancer cell lines, the 3 genes were highly expressed in the ER- MDA-MB-436, MDA-MB-330, MDA-MB-231 and BT-20 cell lines, but not in the ER+ MDA-MB-134-VI and ZR-75-1 cells. In an attempt to characterize the PEA3-group proteins in breast-cancer cells, we first produced and characterized specific antibodies against each of these 3 proteins. The anti-ERM and anti-ER81 antibodies recognized specific strong bands at approximately 72 kDa and 62 kDa, corresponding to ERM and ER81, respectively, in MDA-MB-231 and Hs-578T cells expressing significant levels of the 3 mRNAs. No protein was detected in MCF-7 cells expressing low levels of mRNA for PEA3-group-family genes, or in ZR-75-1 cells, where mRNA was undetectable by Northern blot. Although in vitro-translated PEA3 is specifically immunoprecipitated by anti-PEA3 anti-serum, we were unable to immunoprecipitate PEA3 protein from MDA-MB-231 and Hs-578T cells. In order to study the transcription factor activity of ERM, PEA3 and ER81 proteins in mammary-cancer cells, we tested their ability to transactivate a reporter plasmid containing 3 Ets-binding sites, and were able to show that, in all the breast-cancer cells tested, transfected ERM, PEA3 and ER81 are able to transactivate. Although the target genes of the PEA3 group of transcription factors in breast-cancer cells have yet to be determined, these genes have a potential role in the regulation of growth and the progression of human breast cancer.

Animals↗
PubMed1997

Different points of action of retinoids and anti-estrogens in G1 phase identified in synchronized T-47D breast cancer cells.

Both retinoids and anti-estrogens inhibit breast cancer cell proliferation with accumulation of cells in the G1 phase of the cell cycle, but the effect of retinoids is delayed compared to that of anti-estrogens. To determine whether this temporal difference is due to a simple delay in the action of retinoids on a common site or to different sites of action within the G1 phase, we studied the cell cycle effects of retinoic acid (RA) and the anti-estrogen ICI 164384 (ICI) in T-47D cells partially synchronized by mevalonic acid rescue of lovastatin-induced cell cycle arrest. We found that cells entering the cell cycle semi-synchronously after mevalonic acid rescue of lovastatin treatment were immediately susceptible to ICI but not RA. This suggests that RA may act at a point up-stream and ICI at a point down-stream of lovastatin action. Consistent with this, cells recommencing cell cycle progression after RA treatment were susceptible to the effects of lovastatin, while cells pre-treated with ICI then rescued with estradiol were not. In addition, cells rescued from cell cycle arrest induced by either RA, ICI or lovastatin entered S phase with the same kinetics. Our findings suggest, first, that within G1, RA acts before and ICI acts after the point of lovastatin action and, second, that despite these differences in the initiation of cell cycle arrest, the final nature of the cell cycle arrest is similar. Hence, retinoids and anti-estrogens may be expected to target different cell cycle-regulatory molecules to initiate cell cycle arrest, while overcoming this arrest may be accomplished by the activation of a common molecular pathway.

Cell Cycle↗
PubMed1997

Antiprogestin inhibition of cell cycle progression in T-47D breast cancer cells is accompanied by induction of the cyclin-dependent kinase inhibitor p21.

Progestin antagonists inhibit the proliferation of progesterone receptor-positive cells, including breast cancer cells, by G1 phase-specific actions, but the molecular targets involved are not defined. Reduced phosphorylation of pRB, a substrate for G1 cyclin-dependent kinases (CDKs) in vivo, was apparent after 9 h treatment of T-47D breast cancer cells with the antiprogestins RU 486 or ORG 31710, accompanying changes in S phase fraction. Although the abundance of cyclin D1, Cdk4, and Cdk6 did not decrease cyclin D1-associated kinase activity was reduced by approximately 50% at 9-18 h. Similarly, cyclin E-associated kinase activity decreased by approximately 60% at 12-24 h in the absence of significant changes in the abundance of cyclin E and Cdk2. The CDK inhibitor p21 increased in mRNA and protein abundance and was present at increased levels in cyclin D1 and cyclin E complexes at times when their kinase activity was decreased. Increased p21 protein abundance was observed in another antiprogestin-sensitive cell line, BT 474, but not in two breast cancer cell lines insensitive to antiprogestins. These data suggest increased p21 abundance and concurrent inhibition of CDK activity as a mechanism for antiprogestin induction of growth arrest. Antiprogestin effects on proliferation were markedly reduced after ectopic expression of cyclin D1, indicating that inhibition of cyclin D1 function is a critical element in antiprogestin inhibition of proliferation. However, these data also implicate regulation of cyclin E function in antiprogestin regulation of cell cycle progression.

Breast Neoplasms↗
PubMed1997

Inducible overexpression of cyclin D1 in breast cancer cells reverses the growth-inhibitory effects of antiestrogens.

The development of endocrine resistance in previously sensitive, estrogen receptor-positive breast cancers is a major limitation in the treatment of breast cancer. Because antiestrogens have a cell cycle-specific action on breast cancer cells and influence the expression and activity of several cell cycle-regulatory molecules, the development of aberrant cell cycle control mechanisms is a potential mechanism by which cells might develop resistance to antiestrogens. We postulated that overexpression of cyclin D1, which is a common feature of breast cancer, may confer antiestrogen resistance. We addressed this question in vitro by testing the ability of ectopic cyclin D1 overexpression to overcome the growth-inhibitory effects of tamoxifen and the pure steroidal antiestrogens, ICI 164384 and ICI 182780, in T-47D and MCF-7 human breast cancer cells. In cells stably transfected with a human cyclin D1 cDNA under the control of a metal-inducible metallothionein promoter, cyclin D1 expression was increased 2-4-fold following treatment with zinc. Despite the continued presence of antiestrogen, cyclin D1 induction resulted in the formation of active cyclin D1/Cdk4 complexes, concurrent hyperphosphorylation of the retinoblastoma protein, and entry into S phase of cells previously arrested in G1. Elevated cyclin D1 protein levels were first detected 3 h after treatment with zinc, and the proportion of cells in S phase began to increase 6 h later. The S-phase fraction increased 2-3-fold from 13 to 17% in cells treated with antiestrogen alone, to a peak of 33-38% 15 h after zinc treatment. Both the cyclin D1 protein level and the proportion of cells in S phase increased with increasing concentrations of zinc. We conclude that the ectopic overexpression of cyclin D1 reverses the growth-inhibitory effect of antiestrogens in estrogen receptor-positive breast cancer cells, providing a potential mechanism for clinical antiestrogen resistance.

Antineoplastic Agents↗
PubMed1996

Inducible expression of cyclin D1 in T-47D human breast cancer cells is sufficient for Cdk2 activation and pRB hyperphosphorylation.

The sequential transcriptional activation of cyclins, the regulatory subunits of cell cycle specific kinases, regulates progress through the cell cycle. In mitogen-stimulated cells cyclin D1 induction in early G1 is followed by induction of cyclin E, activation of the cyclin-dependent kinase Cdk2, and hyperphosphorylation of the retinoblastoma gene product (pRB) in mid-to-late G1 phase. T-47D breast cancer cells expressing cyclin D1 under the control of a metal-responsive metallothionein promoter were used to determine whether Cdk2 activation and pRB hyperphosphorylation are consequences of cyclin D1 induction. A 4-5-fold increase in cyclin D1 protein abundance was followed by approximately 2-fold increases in cyclin E protein abundance and Cdk2 activity and by hyperphosphorylation of pRB. These responses were apparent approximately 3 h after the increase in cyclin D1 protein, and approximately 3 h prior to the entry of cyclin D1-stimulated cells into S phase 12 h after zinc treatment. Cyclin D1 immunoprecipitates contained Cdk4 but no detectable Cdk2 and displayed pRb but not histone H1 kinase activity. Cdk2 activation was therefore likely to be due to increased abundance of cyclin E/Cdk2 complexes rather than formation of active cyclin D1/Cdk2 complexes. The sequence of events following zinc induction of cyclin D1 thus mimicked that following mitogen induction of cyclin D1. These data show that cyclin D1 induction is sufficient for Cdk2 activation and pRB hyperphosphorylation in T-47D human breast cancer cells, providing evidence that cyclin D1 induction is a critical event in G1 phase progression.

Breast Neoplasms↗
PubMed1996

Cyclins and breast cancer.

Recent advances in the understanding of cell cycle control by cyclins and cyclin-dependent kinases provide a basis for delineating the molecular mechanisms of proliferation control by steroids and the development and progression of hormone-dependent cancers. Cyclin D1 is necessary, rate-limiting and sufficient for G1 progression in breast cancer cells and regulation of cyclin D1 expression or function is an early response to steroid and steroid antagonist regulation of proliferation. The cyclin D1 gene is amplified in approximately 15%, and its product overexpressed in 40-50%, of primary breast carcinomas. The strong evidence that cyclin D1 plays a major role in cell cycle control in breast epithelial cells suggests that its deregulated expression may have effects on disease progression and phenotype including sensitivity to endocrine therapies.

Breast Neoplasms↗
PubMed1996

Differential effects of retinoids and antiestrogens on cell cycle progression and cell cycle regulatory genes in human breast cancer cells.

Retinoids have antiproliferative effects in human breast cancer cells and share some characteristics with antiestrogens, although the molecular targets involved have yet to be identified in either case. Using T-47D human breast cancer cells, we compared the effects of retinoic acid (RA) and the antiestrogen ICI 164384 on cell cycle phase distribution and the expression of genes with known functions in cell cycle control. Both RA and ICI 164384 inhibited cell cycle progression in G1 phase, but the RA effect was delayed by 16 h. This delay in action was also seen with 9-cis RA and other retinoids. Administration of 17 beta-estradiol abolished the effects of ICI 164384 but was without effect in RA-treated cells. Antiestrogen treatment caused a rapid inhibition of c-myc and cyclin D1 gene expression and reduced Cdk2 activity by more than 50% at 24 h. RA, however, did not affect c-myc or cyclin D1 gene expression, nor did it significantly change the mRNA or protein levels of cyclins D3 or E or cyclin-dependent kinases (CDK) Cdk2 or Cdk4. RA-induced reduction in Cdk2 activity was modest and occurred after %S phase declined, while Cdk4 activity was reduced, coincident with cell cycle changes. However, following either RA or ICI 164384, there was a reduction in the amount of hyperphosphorylated pRB, first apparent well before cell cycle changes were seen. These data demonstrate that: (a) the mechanisms of action of antiestrogens and retinoids are different but converge at pRB; and (b) RA can affect CDK activity without reducing cyclin or CDK levels.

Breast Neoplasms↗
PubMed1996

Cyclin D1 and estrogen receptor messenger RNA levels are positively correlated in primary breast cancer.

The CCND1 gene, encoding the cell cycle regulatory protein cyclin D1, maps to chromosome 11q13, a locus that is amplified in about 13% of breast cancers. Because several studies have indicated a relationship between 11q13 amplification and markers of phenotype including estrogen receptor (ER) status, we tested the relationship between CCND1 and ER gene expression in 364 primary breast cancers using Northern blot analysis. Seventy-three % of samples were positive for ER mRNA, and cyclin D1 mRNA levels in the ER-positive group were significantly higher than those in the ER-negative group (P = 0.0001). When the samples were divided into quartiles of cyclin D1 expression, 58% of samples were ER positive in the lowest quartile and 87% in the highest quartile. The tumors expressing the highest levels of cyclin D1 (7%) were all ER positive. Furthermore, ER mRNA levels in the half with lower cyclin D1 mRNA were significantly less than in the half with higher cyclin D1 levels (P = 0.0001). Using simple regression analysis, there was a significant positive correlation between cyclin D1 and ER mRNA levels in the total population (P = 0.0001). This study demonstrates that cyclin D1 mRNA and ER mRNA are positively correlated in primary breast cancer, but the functional relationship between these genes remains to be elucidated.

Breast Neoplasms↗
PubMed1995

Expression of the cyclin-dependent kinase inhibitors p16INK4, p15INK4B and p21WAF1/CIP1 in human breast cancer.

Aberrant cyclin expression has been implicated in oncogenesis in a number of human cancers. Since altered function of regulators of cyclin-dependent kinase (CDK) activity other than cyclins, in particular CDK inhibitors, might play a similar role in oncogenesis, we examined the expression and regulation of the CDK inhibitors p16INK4, p15INK4B and p21WAF1/CIP1 in human breast cancer cell lines. Both the INK4 and INK4B genes were homozygously deleted in 3 cell lines, while INK4 alone was deleted in 2 cell lines. A further 2 cell lines displayed loss of an allele at this locus, and in 1 of these the remaining allele contained a mis-sense mutation within the coding region of the p16INK4 protein. The majority of cell lines examined, including 2 normal mammary epithelial cell strains, expressed low levels of INK4 mRNA and low or undetectable levels of INK4B mRNA. However, INK4 mRNA was expressed at high levels in 5 cell lines, and this was associated with deletion or inactivation of the retinoblastoma susceptibility gene product pRB but not with mutation of TP53. No deletions of the WAF1/CIP1 gene were observed, but WAF1/CIP1 mRNA levels were reduced in cell lines with TP53 mutation. Transfection of a p16INK4 expression vector into MDA-MB-231 cells lacking the INK4 gene failed to produce any p16INK4-expressing cell lines, suggesting that such cells were selected against in continuous culture. Despite the frequent deletion of INK4 in breast cancer cell lines, no evidence was obtained for INK4 deletions in DNA from 45 primary breast carcinomas. Thus, homozygous deletion of the INK4 gene appears to be a rare event in primary breast cancer.

Base Sequence↗
PubMed1995

Involvement of RB-1, p53, p16INK4 and telomerase in immortalisation of human cells.

Involvement of the retinoblastoma susceptibility (RB-1), p16INK4, p53 and telomerase genes in immortalisation was examined by determining their status in 15 human cell lines representing four immortalisation complementation groups. No abnormalities of RB-1, p53 and p16INK4 were detected in cell lines containing DNA tumour virus proteins known to bind to the protein products of the RB-1 and p53 genes. In contrast, in all other cell lines from each of the four groups either RB-1 was mutant or p16INK4 protein was undetectable and there were cell lines containing p53 mutations in three of the groups. Telomerase activity was detected in 12/15 lines, including some of the virally immortalised lines and in some lines from each group. Since none of these changes correlated with complementation group, other genetic changes must be required for immortalisation.

Base Sequence↗
PubMed1995

Steroidal regulation of cell cycle progression.

Sex steroid hormones and their antagonists have well-defined mitogenic and growth-inhibitory effects on target cells including cancer cells. These effects are mediated by cell cycle phase-specific actions, implying that steroids control rates of cell cycle progression by regulating the expression of key cell cycle regulatory genes. An emerging model of cell cycle control involves transcriptional induction of cyclin genes and consequent activation of cyclin-dependent kinases, which initiate cellular events necessary to complete checkpoints within the cell cycle. Our recent studies have focused on the roles of G1 cyclins, particularly cyclin D1, in the control of cell cycle progression in human breast cancer cells. These studies show that cyclin D1 induction is an early response to mitogenic stimulation by oestrogens and progestins, is rate-limiting for G1 progression and is sufficient for completion of the cell cycle in cells arrested in early G1 phase by serum deprivation. Furthermore, inhibition of cyclin D1 expression is an early response to growth-inhibitory anti-oestrogens. These results suggest that cyclin D1 is a target for regulation of cell cycle progression by sex steroids and their antagonists.

Breast Neoplasms↗
PubMed1995

Antiestrogen inhibition of cell cycle progression in breast cancer cells in associated with inhibition of cyclin-dependent kinase activity and decreased retinoblastoma protein phosphorylation.

To define the mechanisms by which antiestrogens inhibit breast cancer cell proliferation, the effects of the antiestrogen ICI 182780 on G1 cyclins and their cyclin-dependent kinase (CDK) partners were investigated in MCF-7 cells. Inhibition of entry into S phase became evident 9 h after treatment, with the proportion of cells in S phase reaching a minimum by 24 h. ICI 182780 increased the proportion of the hypophosphorylated, growth inhibitory form of the retinoblastoma protein (pRB). This change began at 4-6 h, preceding effects on S phase. This suggests that there are early effects on the activities of CDKs that target pRB that are not merely a consequence of changes in cell cycle progression. The kinase activity of Cdk2 decreased to low levels at 18-24 h when changes in S phase and pRB phosphorylation were well advanced. An earlier effect was seen on kinase activity associated with immunoprecipitated cyclin D1, which was reduced approximately 40% by 12 h, with further decreases at 18-24 h. Cdk2 and Cdk4 protein levels remained constant over 24 h. Cyclin D1 messenger RNA and protein were down-regulated by ICI 182780 from 2 h, with levels halved at 8 h. ICI 182780 also increased the expression of the CDK inhibitors p27KIP1 and p21WAF1/CIP1 at later times. These observations are compatible with the hypothesis that antiestrogens block entry of cells into S phase and inhibit cell proliferation as the consequence of an early decline in pRB phosphorylation contributed to by reduced cyclin D1/Cdk4 activity. At later times, increased CDK inhibitor abundance may act to repress Cdk2 and Cdk4 activities, causing additional reductions in pRB phosphorylation, thus maintaining the antiestrogen blockade of cell cycle progression.

Breast Neoplasms↗
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↗
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

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↗
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↗