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R L Sutherland

Publications and source records attributed to R L Sutherland.

At least 55 records · Page 3Linked to original sources
PubMed1998

Cyclin D1 protein is overexpressed in hyperplasia and intraductal carcinoma of the breast.

The cell cycle regulatory gene cyclin D1 is a candidate oncogene in breast cancer. It is overexpressed in 30-50% of invasive primary breast cancers and plays a key role in mediating mitogenic responses to steroids and growth factors in breast cancer cells in vitro. Because the role of cyclin D1 in the proliferative and early noninvasive stages of breast cancer is largely unknown, we examined normal breast epithelium (NBE), proliferative disease (PD), ductal carcinoma in situ (DCIS), and invasive carcinoma (IC) to evaluate the timing and possible importance of cyclin D1 expression in the development of breast cancer. Using immunohistochemistry, we examined cyclin D1 protein expression in 471 breast tissue samples. A quantitative scoring system for immunohistochemistry based on percentage of positive cells was developed that correlated with Western blot analysis of antigen concentration in paired samples (r2 = 0.91, P = 0.003). A sample was considered positive if >5% of relevant epithelial cells demonstrated nuclear staining. Cyclin D1 positivity was observed in 11.7% (7 of 60) samples of NBE, 25% (11 of 44) of PD without atypia, 39.4% (13 of 33) of atypical ductal hyperplasia, 43.6% (17 of 39) of low-grade DCIS, 47.9% (23 of 48) of high-grade DCIS, and 48.3% (99 of 205) of IC. Cyclin D1 expression was significantly higher in PD than NBE (P = 0.006) and in DCIS than PD (P = 0.038). There was no significant increase from DCIS to IC (P = 0.52). The increase in cyclin D1 expression in the overall progression from NBE to IC was also highly significant (P = 0.0001). Therefore, cyclin D1 expression was detected at levels significantly greater than in NBE in the earliest proliferative epithelial lesions of the breast with a further significant increase accompanying the progression to any form of cancer. This suggests that overexpression of cyclin D1 protein is important at the earliest stages of breast oncogenesis and continues to have a crucial role throughout the development of malignancy.

Breast Neoplasms↗
PubMed1997

Differential phosphorylation of T-47D human breast cancer cell substrates by D1-, D3-, E-, and A-type cyclin-CDK complexes.

The cyclin-dependent kinases (CDKs) promote cell cycle transitions in mammalian cells by phosphorylation of key substrates. To characterize substrates of the G1 and S phase cyclin-CDK complexes, including cyclin D1-CDK4, cyclin D3-CDK4, cyclin D3-CDK6, cyclin E-CDK2, and cyclin A-CDK2, which are largely undefined, we phosphorylated T-47D breast cancer cell nuclear lysates partially purified by ion-exchange chromatography with purified baculovirus expressed cyclin-CDK complexes. A comparison of the substrates that were phosphorylated by the different cyclin D-CDKs revealed some common as well as specific substrates. Hence, cyclin D1-CDK4 specifically phosphorylated a 38-kDa protein while cyclin D3-CDK4 specifically phosphorylated proteins of 105, 102, and 42 kDa. A 24-kDa protein was phosphorylated by both complexes. Cyclin D3-CDK6 exhibited similar substrate preferences to cyclin D3-CDK4, phosphorylating the 105- and 102-kDa proteins but not the 24-kDa protein. Hence, both the cyclin D1 and D3 as well as CDK4 and CDK6 subunits can confer substrate specificity on the overall cyclin D-CDK complex. Cyclin E-CDK2 and cyclin A-CDK2 phosphorylated a greater number of substrates than the cyclin D-CDKs, ranging in size from 10 kDa to over 200 kDa. Twenty-two substrates were common to both complexes, while six were specific for cyclin A-CDK2 and only one protein of 34 kDa was specific for cyclin E-CDK2. These studies indicate that cyclins E and A modulate the specificity of CDK2 and have demonstrated substrates that may be important for the specific roles of these cyclin-CDKs during G1 and S phase progression. Protein sequencing of one of the cyclin-CDK substrates characterized in this study identified this protein as nucleolin, a previously characterized CDC2 (CDK1) substrate, thus indicating the utility of this approach in identifying cyclin-CDK targets. These results show that both the cyclin and CDK subunits can regulate the substrate specificity of the overall cyclin-CDK complex and have demonstrated numerous substrates of D-, E-, and A-type cyclin-CDK complexes potentially involved in regulating transit through the G1 and S phases of the cell cycle.

Amino Acid Sequence↗
PubMed1997

EMS1 amplification can occur independently of CCND1 or INT-2 amplification at 11q13 and may identify different phenotypes in primary breast cancer.

Chromosome 11q13 is amplified in about 13% of primary breast cancers. CCND1, encoding the cell cycle regulatory gene cyclin D1, and EMS1, encoding a filamentous actin binding protein, are favoured candidate onocogenes, whereas INT-2 is an unexpressed gene at this locus. In this study we tested the possibility that different regions of this large amplicon could be independently amplified and subsequently defined the phenotype of EMS1 amplified tumours in a series of 961 primary breast carcinomas. Using DNA slot blots, EMS1 was amplified in 15.2% of samples: 5.4% were coamplified for CCND1; 7.9% coamplified for INT-2 and 6.7% showed EMS1 amplification alone. The degree of amplification of CCND1 and INT-2 was highly correlated (P =0.0001). In contrast, no such relationship existed between EMS1 and CCND1 or INT-2 amplification, demonstrating independent amplification of EMS1 in 44% of amplified tumours. EMS1 amplification (> or = twofold increase in copy number) was positively correlated with patient age > or = 50 years (P = 0.025), ER positivity (P = 0.022), PgR positivity (P = 0.018), and was negatively correlated with HER-2/neu (c-erbB2) amplification (P = 0.01). In common with CCND1/INT-2, EMS1 amplification was associated with increased risk of relapse in patients with lymph node-negative disease (P = 0.028). In contrast, EMS1 and CCND1/INT-2 amplification appeared to confer different phenotypes in ER positive and negative tumours. A > or = threefold increase in EMS1 copy number was associated with an apparent increased risk of relapse and death in patients with ER negative tumours, but was without effect in ER positive tumours. In contrast, CCND1/INT-2 amplification had no effect in the patients with ER negative tumours but was associated with early relapse in ER positive patients. Thus EMS1 amplification may identify subgroups of breast cancer patients with increased probability of relapse and death distinct from those identified by CCND1/INT-2 amplification. Further studies are required to more clearly determine the functional consequences of EMS1 overexpression and a biological basis for the relationship between EMS1 amplification and phenotype in breast cancer.

Breast Neoplasms↗
PubMed1997

Estrogen-induced activation of Cdk4 and Cdk2 during G1-S phase progression is accompanied by increased cyclin D1 expression and decreased cyclin-dependent kinase inhibitor association with cyclin E-Cdk2.

Estrogens induce cell proliferation in target tissues by stimulating progression through G1 phase of the cell cycle, but the underlying molecular targets remain undefined. To determine the role of the cyclin/cyclin-dependent kinase (CDK)/retinoblastoma protein (pRB) pathway in this response we treated MCF-7 breast cancer cells with the pure estrogen antagonist ICI 182780 to inhibit estrogen-induced gene expression and induce G1 phase arrest. Subsequent treatment with 17beta-estradiol resulted in the synchronous entry of cells into S phase commencing at 12 h. The proportion of cells in S phase reached a maximum of 60% at 21-24 h. Cells subsequently completed mitosis and entered a second semisynchronous round of replication. Entry into S phase was preceded by increased activity of both Cdk4 and cyclin E-Cdk2 and hyperphosphorylation of pRB, all within the first 3-6 h of estradiol treatment. The increase in Cdk4 activity was accompanied by increases in cyclin D1 mRNA and protein, indicating that an initiating event in the activation of Cdk4 was increased cyclin D1 gene expression. In contrast, the levels of Cdk2 and the CDK inhibitors p21 (WAF1/CIP1/SDI1) and p27 (KIP1) in total cell lysates and in cyclin E immunoprecipitates were unaltered at these early time points. However, an inhibitory activity was present in antiestrogen-pretreated cell lysates toward recombinant cyclin E-Cdk2 and was relieved by estradiol treatment. This activity was attributable predominantly to p21. These apparently conflicting data were resolved by performing gel filtration chromatography, which revealed that only a minority of cyclin E-Cdk2 complexes were active following estradiol treatment. Active complexes eluted at a higher molecular weight than inactive complexes, were relatively deficient in both p21 and p27, and contained Cdk2 with increased threonine 160 phosphorylation, consistent with a mechanism of activation of cyclin E-Cdk2 involving both reduced CDK inhibitor association and CDK-activating kinase-mediated phosphorylation of Cdk2. These results provide an explanation for the early activation of both cyclin D1-Cdk4 and cyclin E-Cdk2 complexes that accompany G1-S phase progression in response to estradiol.

Breast Neoplasms↗
PubMed1997

Structural determinants of the interaction between the erbB2 receptor and the Src homology 2 domain of Grb7.

The Src homology 2 (SH2) domain-containing protein Grb7 and the erbB2 receptor tyrosine kinase are overexpressed in a subset of human breast cancers. They also co-immunoprecipitate from cell lysates and associate directly in vitro. Whereas the Grb7 SH2 domain binds strongly to erbB2, the SH2 domain of Grb14, a protein closely related to Grb7, does not. We have investigated the preferred binding site of Grb7 within the erbB2 intracellular domain and the SH2 domain residues that determine the high affinity of Grb7 compared with Grb14 for this site. Phosphopeptide competition and site-directed mutagenesis revealed that Tyr-1139 of erbB2 is the major binding site for the Grb7 SH2 domain, indicating an overlap in binding specificity between the Grb7 and Grb2 SH2 domains. Substituting individual amino acids in the Grb14 SH2 domain with the corresponding residues from Grb7 demonstrated that a Gln to Leu change at the betaD6 position imparted high affinity erbB2 interaction, paralleled by a marked increase in affinity for the Tyr-1139 phosphopeptide. The reverse switch at the betaD6 position abrogated Grb7 binding to erbB2. This residue therefore represents an important determinant of SH2 domain specificity within the Grb7 family.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Cyclin D2 activates Cdk2 in preference to Cdk4 in human breast epithelial cells.

To investigate the possibility of differing roles for cyclins D1 and D2 in breast epithelial cells, we examined the expression, cell cycle regulation and activity of these two G1 cyclins in both 184 normal breast epithelial cells and T-47D breast cancer cells. Synchronisation studies in 184 cells demonstrated that cyclin D1 and cyclin D2 were differentially regulated during G1, with cyclin D2 abundance increasing by 3.7-fold but only small changes in cyclin D1 abundance observed. The functional consequences of increased cyclin D2 expression were examined in T-47D cells, which express no detectable cyclin D2. Induced expression of cyclin D2 resulted in increases in cyclin E expression, pRB phosphorylation and the percentage of cells in S-phase, while constitutive expression resulted in a consistent trend toward reduced dependence on serum for continued proliferation. Thus, cyclin D2 is a positive regulator of G1 progression in breast cells analogous to the well-documented effects of cyclin D1. Indeed, equimolar concentrations of inducible cyclin D1 and D2 resulted in quantitatively similar cell cycle effects. Marked divergence was found, however, in the CDKs activated by the two cyclins in breast epithelial cells. Cyclin D2 complexes contained a higher Cdk2/Cdk4 ratio than cyclin D1 complexes. The cyclin D2-associated kinase activity was largely inhibited by Cdk2-specific inhibitors and could phosphorylate histone H1, a substrate for Cdk2 but not for Cdk4 and Cdk6. Therefore, cyclin D2 preferentially activated Cdk2 in breast epithelial cells. In contrast, Cdk4 and Cdk6 were predominantly responsible for cyclin D1-associated kinase activity as previously reported. Thus, although cyclins D1 and D2 elicited similar effects on breast epithelial cell cycle progression they appeared to achieve this end via activation of different CDKs. This is the first evidence of cyclin D2 activating Cdk2 in mammalian cells thus providing further evidence that D-type cyclins are not necessarily redundant.

Breast↗
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

Expression and function of members of the cytokine receptor superfamily on breast cancer cells.

Receptors for the cytokines leukemia inhibitory factor (LIF), interleukin-6 (IL-6), oncostatin M (OSM), ciliary neurotrophic factor (CNTF) and interleukin-11 (IL-11) are members of the structurally conserved hemopoietin receptor superfamily. In addition, they all share the transmembrane signalling protein gp130. In this paper the expression and function of this family of receptors in breast cancer cells was examined. RT-PCR analyses demonstrated that gp130 was expressed in 12/12 breast cell lines and the specific receptor alpha-chains for IL-6, LIF, IL-11 and CNTF were expressed in the majority of these cell lines. This was in contrast to other hemopoietin receptors. Examination of 50 clinical samples of malignant breast tissue by RT-PCR showed a similar pattern of expression of gp130 associated receptors. Treatment of breast cancer cell lines with OSM resulted in changes in cellular morphology. Cellular proliferation was inhibited following exposure to OSM (3/4 cell lines), IL-11 (2/4 cell lines), and by IL-6 and LIF (1/4 cell lines). Cell surface binding of LIF and OSM was also documented. The expression of these receptors in 12/12 cell lines and greater than 95% of clinical samples suggests that these molecules may be important in regulating the growth of breast cells.

Base Sequence↗
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

Coexpression and cross-regulation of the prolactin receptor and sex steroid hormone receptors in breast cancer.

The sex steroid hormones and PRL interact synergistically to control the neoplastic growth of the mammary gland. The basis for this hormonal synergy is unknown, but may involve cellular coexpression of the sex steroid and PRL receptors, coupled with receptor cross-regulation. To examine this hypothesis the expression of the sex steroid and PRL receptors was examined in 20 human breast cancer cell lines and 123 primary breast cancers. Regulation of sex steroid receptors by PRL and of the PRL receptor by sex steroids was examined in T-47D and MCF-7 breast cancer cells. Northern analysis of the breast cancer cell lines and tumors indicated that the PRL receptor and the sex steroid receptors were coexpressed. The level of PRL receptor expression in the breast cancer cell lines was linearly related to that of the estrogen and progesterone receptors, but not to that of the androgen receptor. In MCF-7 and T-47D cells, acute treatment with progestins and androgens and long term treatment with estrogens increased PRL receptor levels. Analysis of sex steroid receptor messenger ribonucleic acid and binding activity showed that acute PRL treatment produced a time- and concentration-dependent increase in progesterone receptor and a decrease in androgen receptor. These results indicate that receptors for sex steroids and PRL are coexpressed and are cross-regulated, providing a potential mechanism for the observed synergy among estrogen, progesterone, and PRL in the control of tumor growth.

Blotting, Northern↗
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

Identification of PRG1, a novel progestin-responsive gene with sequence homology to 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

To define early molecular targets of progestin action, the differential display technique was used to identify genes with altered levels of expression in T-47D breast cancer cells treated with the synthetic progestin ORG 2058 for 3 h. PRG1 was first isolated as a 200-bp cDNA clone and its progestin regulation confirmed by Northern analysis. Cloning of the complete coding region of PRG1 revealed that it shared a high degree of amino acid sequence identity with isoforms of the enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from several tissues and species. Expression of PRG1 mRNA was observed in several normal breast epithelial and breast cancer cell lines and in a variety of human tissues, with highest expression in the breast, aorta, and brain. In T-47D cells, PRG1 mRNA was rapidly and transiently induced by progestins, expression peaking between 2 and 4 h and returning to control levels by 12 h. Progestin-induced increases in PRG1 mRNA were inhibited by the progestin antagonist RU 486 and occurred via the progesterone receptor. Progestin induction of PRG1 mRNA was also inhibited by actinomycin D but not by cycloheximide. PRG1 is therefore a novel human gene that is directly regulated by progestins via the progesterone receptor.

Amino Acid Sequence↗
PubMed1997

Antisense estrogen receptor RNA expression increases epidermal growth factor receptor gene expression in breast cancer cells.

In human breast cancer, progression to a more malignant phenotype is often accompanied by decreased expression of estrogen receptor (ER) and increased expression of epidermal growth factor receptor (EGFR). Higher levels of this receptor tyrosine kinase are found in tumors lacking ER, and a quantitative, inverse relationship exists between the level of ER and EGFR mRNA in human breast cell lines. Antisense ER (ASER) RNA was used to evaluate the consequence of decreased ER expression in breast cancer cells, specifically to determine whether ER is involved in the regulation of EGFR gene expression. ER-positive MCF-7 human breast cancer cells were transfected with ASER, and clones constitutively expressing ASER RNA had decreased ER and up to a 3-fold increase in the expression of EGFR mRNA. To confirm that this observation was a direct consequence of ASER expression, a metal-inducible ASER expression construct was transfected into MCF-7 cells, and transfected clones were isolated and characterized. Northern analysis revealed an induction of ASER RNA within 1 h of the addition of zinc, which was followed by a 4-fold increase in EGFR mRNA levels, maximal at 6-12 h. The basal level of expression of the glucocorticoid receptor is also inversely related to that of ER among breast cancer cell lines, but neither constitutive nor inducible expression of ASER affected the expression of glucocorticoid receptor. These data support the hypothesis that the level of expression of ER specifically influences the expression of EGFR in human breast cancer cells and provides a potential link between loss of steroid sensitivity and the acquisition of autonomous growth.

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

Expression and tyrosine phosphorylation of EMS1 in human breast cancer cell lines.

The EMS1 gene encodes an 80/85 kDa c-src substrate and localises with the CCND1 gene to chromosome 11q13. This locus is amplified in approximately 13% of human breast cancers. EMS1 gene amplification and expression were characterised in a panel of human breast cancer cell lines to determine at what levels expression is regulated. The degree of tyrosine phosphorylation of EMS1 protein was also determined and compared with the activity of src-family kinases. The EMS1 gene was amplified in 6 of 20 cell lines investigated: MDA-MB-134, -157, -175, -453, ZR-75-1 and MCF-7. In the MDA-MB-157 and MCF-7 cell lines, EMS1 was amplified in the absence of CCND1 gene amplification. EMS1 protein levels were increased relative to normal breast epithelial cells in 6 cell lines (ZR-75-1, MDA-MB-134, -175, 453, MCF-7 and BT-474). Of these, BT-474 is the only cell line that does not exhibit EMS1 amplification or increased EMS1 mRNA levels. EMS1 tyrosine phosphorylation was 3-fold higher in BT-474 and T-47D cells, which exhibited relatively high total src activity coupled with expression of both c-fyn and c-yes, than in MDA-MB-453 cells, which expressed only c-yes. Our results therefore demonstrate gene amplification to be the predominant mechanism underlying EMS1 over-expression in human breast cancer cell lines and identify tyrosine phosphorylation as a further level at which regulation of this protein may be perturbed.

Breast Neoplasms↗
PubMed1996

High activity, soluble, bacterially expressed human vitamin D receptor and its ligand binding domain.

The effects of 1 alpha, 25(OH)2 vitamin D3 on cell growth and differentiation are primarily mediated by the nuclear vitamin D receptor (VDR). In order to study aspects of receptor function and ultimately the structural basis of the VDR-ligand interaction, it is necessary to produce large quantities of purified VDR. To achieve this, we have expressed the human VDR and its ligand binding domain in E. coli as fusion proteins with the maltose binding protein using the expression vector pMal-c2. In this system high level expression of both fusion proteins in a soluble form was achieved, whereas previous attempts to express the VDR in E. coli have resulted in an insoluble product. After affinity purification on amylose resin, the fusion proteins were isolated with yields of 10-20 mg/l of culture. Both forms of the recombinant receptor bound 1 alpha, 25(OH)2 vitamin D3 with high affinity; estimated Kd values from Scatchard analysis for the purified full-length receptor and the ligand binding domain were 0.16 +/- 0.07 nM and 0.04 +/- 0.02 nM, respectively. The nonhypercalcemic analogs of vitamin D, MC903 and delta 22-1, 25S, 26 (OH)3 vitamin D3, bound the recombinant fusion proteins with a similar affinity to the native ligand, 1 alpha, 25(OH)2 vitamin D3. In addition, the full-length VDR fusion protein was shown by gel shift analysis to bind weakly to the human osteocalcin gene vitamin D response element, an interaction greatly facilitated by addition of RXR alpha. These results show that the bacterial expression system detailed here is readily able to produce soluble and functional VDR and its ligand binding domain in high yield. These proteins are easily purified and should be suitable for further structural and functional analysis.

Binding, Competitive↗