Biomedical subjects

L C Murphy

Publications and source records attributed to L C Murphy.

At least 19 recordsLinked to original sources

PubMed2000

Altered expression of estrogen receptor coregulators during human breast tumorigenesis.

The hypothesis that altered expression of specific coactivators/repressors of the estrogen receptor occurs during human breast tumorigenesis in vivo is examined in this study. Using in situ hybridization and reverse transcription-PCR assays, the expression of two coactivators (SRA and AIB1) and one repressor (REA) of the estrogen receptor was compared between matched breast tumors and adjacent normal human breast tissue. The levels of SRA and AIB1 mRNA were increased in tumors compared with normal tissues (n = 19; Wilcoxon matched pairs test; P < 0.01). In contrast, the expression of REA mRNA was not different between tumors and normal tissues (n = 19; Wilcoxon; P = 0.110). The ratios of AIB1:REA and SRA:REA were higher (Wilcoxon; P < 0.05) in tumors compared with normal tissues. Furthermore, SRA:AIB1 was higher (Wilcoxon; P = 0.0058) in tumors compared with normal tissues. Although our study is small, these data are consistent with the above hypothesis and suggest that such alterations may have a role in the altered estrogen action occurring during breast tumorigenesis.

Breast↗

PubMed2000

Expression of a repressor of estrogen receptor activity in human breast tumors: relationship to some known prognostic markers.

The expression of a specific repressor of estrogen receptor activity (REA) was investigated by a semiquantitative reverse transcription-PCR assay in 40 human breast tumor biopsy samples with respect to steroid hormone receptor status and other known prognostic variables. The data showed that REA expression was positively correlated with estrogen receptor (ER) levels as defined by ligand-binding assays (Spearman r = 03231; P = 0.042) and that the median level of REA mRNA was significantly (Mann-Whitney two-tailed test, P = 0.0424) higher in ER+ tumors (median = 94.5; n = 30) compared with ER- tumors (median = 645; n = 10), with no significant differences (P = 0.4988) associated with progesterone receptor status alone. In addition, REA expression was inversely correlated with tumor grade (Spearman r = -0.4375; P = 0.0054). When the tumors were divided into two groups based on grade, REA expression was significantly (Mann-Whitney two-tailed test, P = 0.0024) higher in low-grade (median = 97; n = 16) compared with high-grade (median = 76; n = 23) tumors. These results provide preliminary data suggesting that the expression of REA varies among breast tumors and is correlated with known treatment response markers and inversely correlated with a marker of breast cancer progression. REA together with ER status may be an improved marker of endocrine therapy responsiveness in human breast cancer.

Biomarkers, Tumor↗

PubMed2000

Lumican and decorin are differentially expressed in human breast carcinoma.

Previous studies have shown that lumican is expressed and increased in the stroma of breast tumours. Lumican expression has now been examined relative to other members of the small leucine-rich proteoglycan gene family in normal and neoplastic breast tissues, to begin to determine its role in breast tumour progression. Western blot study showed that lumican protein is highly abundant relative to decorin, while biglycan and fibromodulin are only detected occasionally in breast tissues (n=15 cases). Further analysis of lumican and decorin expression performed in matched normal and tumour tissues by in situ hybridization showed that both mRNAs were expressed by similar fibroblast-like cells adjacent to epithelium. However, lumican mRNA expression was significantly increased in tumours (n=34, p<0.0001), while decorin mRNA was decreased (p=0.0002) in neoplastic relative to adjacent normal stroma. This was accompanied by a significant increase in lumican protein (n=12, p=0.0122), but not decorin. Further evidence of altered lumican expression in breast cancer was manifested by discordance between lumican mRNA and protein localization in some regions of tumours but not in adjacent morphologically normal tissues. It is concluded that lumican is the most abundant of these proteoglycans in breast tumours and that lumican and decorin are inversely regulated in association with breast tumourigenesis.

Adult↗

PubMed2000

Functional characteristics of a novel murine estrogen receptor-beta isoform, estrogen receptor-beta 2.

We have isolated a highly expressed splice variant mRNA of murine estrogen receptor-beta (ERbeta), mERbeta2, containing an in-frame 54 nucleotide insertion between exons 5 and 6 of wild-type mERbeta1. The predicted ERbeta2 protein contains 18 amino acids inserted in the ligand binding domain of mERbeta1. Recombinant protein generated by in vitro transcription/translation showed that mERbeta2 had markedly reduced ligand binding (K(D)=17.7+/-4.7 nM, mean+/-s.e.m., n=3) compared with mERbeta1-bound (3)H-estradiol (K(D)=0.56+/- 0.19 nM, mean+/-s.e.m., n=3). Both receptors bound similarly to palindromic estrogen responsive elements (EREs) in vitro and in vivo, and similarly bent DNA. Transcriptional activity was assessed using transient transfection analysis into a homologous murine cell line, NIH 3T3 cells. mERbeta1 transactivated ERE-tk-CAT reporter genes similarly to mERalpha, whereas mERbeta2 had little activity except at high ligand concentrations. However, under conditions in which mERbeta2 is unlikely to be ligand saturated, co-transfected mERbeta2 inhibited activity of mERalpha and possibly mERbeta1 on ERE-tk-CAT genes. Using a 'novel raloxifene responsive' gene reporter system (TGF-beta3-CAT), we found the ability of estradiol and LY117018 to activate both mERalpha and mERbeta1 on this promoter was identical, and mERbeta2 activity in the presence of either estradiol or LY117018 was only slightly less than that observed with either mERbeta1 or mERalpha. Both mERbeta1 and mERbeta2 when liganded with LY117018 inhibited transcription at a classical ERE-regulated promoter under these transfection conditions, which was in marked contrast to their stimulatory effect at the transforming growth factor-beta3 promoter. These data suggest that responsiveness of gene expression to a relatively highly expressed variant murine ERbeta isoform, mERbeta2, is both ligand and promoter specific. Determination of the relative level of expression of mERbeta1 mRNA and mERbeta2 mRNA in mouse tissues indicated predominance of mERbeta2 mRNA in some but not all tissues. These data suggest that the mERbeta2 may have some tissue-specific and promoter-specific modulatory effects.

3T3 Cells↗

PubMed1999

Estrogen receptor-alpha mRNA variants in murine and human tissues.

A side-by-side comparison of several normal mouse and human tissues was undertaken in order to determine if exon-deleted variant ER-alpha mRNAs are expressed in the mouse. The data showed that the complex pattern of ER-alpha alternative splicing that is detected in multiple human tissues was not apparent in murine tissues. Only low levels of an exon-4 deleted ER-alpha transcript were detected in murine tissues, although multiple relatively abundant exon-deleted ER-alpha transcripts were detected in human tissues. The data support a species-specific difference in the expression of ER-alpha variant mRNAs between mouse and human.

Alternative Splicing↗

PubMed1999

Expression of the steroid receptor RNA activator in human breast tumors.

The expression of the recently described steroid receptor RNA activator (SRA) was measured by semiquantitative reverse transcription-PCR within 27 independent breast tumors, spanning a wide spectrum of grade and estrogen receptor (ER) and progesterone receptor (PR) levels. Subgroup analysis showed that SRA expression was similar in ER+/PR+ (median = 65.5, n = 8) and in ER-/PR- (median = 94.6, n = 5) tumors. Interestingly, SRA expression in these two subgroups was significantly (Mann-Whitney rank-sum test, P < 0.05) lower than that observed in ER+/PR- (median = 156.4, n = 6) and ER-/PR+ (median = 144.8, n = 8) tumors. A variant form of SRA, presenting a deletion of 203 bp within the SRA core sequence, was also observed in breast tumor tissues. The relative expression of this new SRA isoform correlated with tumor grade (Spearman coefficient r = 0.53, n = 27, P = 0.004). These data suggest that changes in the expression of SRA-related molecules occur during breast tumor progression.

Breast Neoplasms↗

PubMed1999

Expression of estrogen receptor beta1, beta2, and beta5 messenger RNAs in human breast tissue.

A triple-primer PCR assay was developed, based on the coamplification of estrogen receptor (ER)-beta1, -beta2, and -beta5 cDNAs, to investigate the relative expressions of the corresponding mRNAs in breast cancer lines and in 53 independent breast tumors. The expression of ER-beta2 and ER-beta5 mRNAs was higher than that of ER-beta1 mRNA in both cancer cell lines and breast tumors. In breast tumors, increases in the ER-beta2:ER-beta1 and ER-beta5:ER-beta1 mRNA expression ratios were observed, which positively correlated with the level of tumor inflammation and tumor grade, respectively. A trend toward an increase of these ratios was also found in tumors, as compared to the normal adjacent breast tissue available for 13 cases. Our data suggest that changes in the relative expression of ER-beta1, -beta2, and -beta5 mRNAs occur during breast tumorigenesis and tumor progression.

Adult↗

PubMed1999

Estrogen receptor-beta messenger RNA expression in human breast tumor biopsies: relationship to steroid receptor status and regulation by progestins.

When the level of estrogen receptor (ER)-beta mRNA in tumors, determined by reverse transcription-PCR, was assessed according to either ER status or PR status alone, determined by ligand binding assays, the level of ER-beta mRNA was significantly lower in PR+ tumors compared with PR- tumors (P = 0.036), and no association with ER status was found. Subgroup analysis showed that ER-beta mRNA expression in ER+/PR+ breast tumors was significantly less than in ER+/PR- (P = 0.009), ER-/PR+ (P = 0.029), and ER-/PR- (P = 0.023) groups. Interestingly, the ER-beta mRNA expression was specifically decreased by progestin in T47D breast cancer cells. The data suggest the possibility that expression of ER-beta in human breast tumors is a marker of endocrine therapy responsiveness.

Biopsy↗

PubMed1999

Mammaglobin, a potential marker of breast cancer nodal metastasis.

The Mammaglobin gene, a breast-specific member of the uteroglobin gene family, has been previously identified as being overexpressed in some breast tumours, but the cellular origin and relationship to tumour progression are unknown. Using a subtractive hybridization approach, mammaglobin mRNA has also been found to be overexpressed in the in situ compared to the invasive element within an individual breast tumour. Further study by in situ hybridization performed in 13 breast tumours, selected to include normal, in situ, and invasive primary tumour elements, and in most cases axillary lymph node metastases, revealed that mammaglobin expression occurs in all elements, is restricted to epithelial cells, and is significantly increased in tumour cells compared with normal cells ( p< 0.04). Analysis of mammaglobin expression within 20 independent primary breast tumours and their corresponding axillary lymph nodes revealed that all 13 lymph nodes positive and none of the seven nodes negative for metastatic breast carcinoma by histology were mammaglobin-positive by reverse transcription-polymerase chain reaction (RT-PCR) ( p=0.0001). These results suggest that mammaglobin could be a marker of axillary lymph node breast metastases.

Adult↗

PubMed1999

Psoriasin (S100A7) expression and invasive breast cancer.

Alteration of psoriasin (S100A7) expression has previously been identified in association with the transition from preinvasive to invasive breast cancer. In this study we have examined persistence of psoriasin mRNA and protein expression in relation to prognostic factors in a cohort of 57 invasive breast tumors, comprising 34 invasive ductal carcinomas and 23 other invasive tumor types (lobular, mucinous, medullary, tubular). We first developed an IgY polyclonal chicken antibody and confirmed specificity for psoriasin by Western blot in transfected cells and tumors. The protein was localized by immunohistochemistry predominantly to epithelial cells, with both nuclear and cytoplasmic staining, as well as occasional stromal cells in psoriatic skin and breast tumors; however, in situ hybridization showed that psoriasin mRNA expression was restricted to epithelial cells. In breast tumors, higher levels of psoriasin measured by reverse transcriptase-polymerase chain reaction and Western blot (93% concordance) were significantly associated with estrogen and progesterone receptor-negative status (P < 0.0001, P = 0.0003), and with nodal metastasis in invasive ductal tumors (P = 0. 035), but not with tumor type or grade. Psoriasin expression also correlated with inflammatory infiltrates (all tumors excluding medullary, P = 0.0022). These results suggest that psoriasin may be a marker of aggressive behavior in invasive tumors and are consistent with a function as a chemotactic factor.

Blotting, Western↗

PubMed1999

Influence of estrogen receptor variants on the determination of ER status in human breast cancer.

Determination of estrogen receptor alpha (ER) status in breast cancer is an important predictive factor for clinical response to endocrine therapy. We have recently shown that discrepancies in ER status determined by immunohistochemical assay (ER-IHA) can occur between amino-terminal (1D5) and carboxyl-terminal (AER-311) targeted ER antibodies and that those tumors which demonstrate discordance are associated with increased expression of truncated ER variant mRNAs. In this study, we have explored this observation to examine if ER variant expression can exert a direct effect on ER-IHA or whether this association is attributable to the characteristics of the antibodies. ER negative cos-1 cells were transfected with expression vectors containing wild type ER (wt-ER) and/or a frequently expressed truncated variant, ER-clone-4 variant. We found that ER-IHA performed with the same N- and C-terminal targeting ER antibodies on cos-1 cells expressing wt-ER alone demonstrated no difference in signals by western blot (P > 0.1). However, co-expression of wt-ER and the truncated ER-clone-4 variant, resulted in discordant IHA results with relatively higher ER-IHA H-scores from N-terminal antibodies (P < 0.03). Furthermore, re-examination of a subset of breast tumors previously studied by ER-IHA showed persistent concordance in 4/5 cases and persistent differences in 3/5 cases with a different pair of ER antibodies. We conclude that the presence of truncated ER variant proteins can interfere with the interpretation of ER status determined by IHA and that this may account for some of the inconsistencies between ER status and response to endocrine therapy.

Antibodies, Neoplasm↗

PubMed1999

Oestrogen receptor-alpha variant mRNA expression in primary human breast tumours and matched lymph node metastases.

We have shown previously that the relative expression of a truncated oestrogen receptor-alpha variant mRNA (ER clone 4) is significantly increased in axillary node-positive primary breast tumours compared with node-negative tumours. In this study, we have examined the relative expression of clone 4-truncated, exon 5-deleted and exon 7-deleted oestrogen receptor-alpha variant mRNAs in 15 primary breast tumour samples and in synchronous axillary lymph node metastases. Overall, there were no significant differences between the primary tumours and the matched metastases in the relative expression of these three specific variant mRNAs. Furthermore, the pattern of all deleted oestrogen receptor-alpha variant mRNAs appeared conserved between any primary and its matched secondary tumour.

Breast Neoplasms↗

PubMed1999

Altered expression of exon 6 deleted progesterone receptor variant mRNA between normal human breast and breast tumour tissues.

The progesterone receptor (PR) is an important prognostic marker in breast cancer as well as a marker of responsiveness to endocrine therapies. The presence of several exon-deleted PR variant mRNAs in both normal and neoplastic breast samples has recently been reported. Amongst them, a variant mRNA deleted in exon 6 (D6-PR mRNA) that if translated would encode a truncated PR-like protein missing the hormone binding domain and one of the transactivating domains of the wild-type PR protein. In order to determine whether changes in D6-PR variant expression could occur during tumorigenesis, its expression was investigated by reverse transcription and polymerase chain reaction in ten normal reduction mammoplasty samples, nine breast tumours with high PR levels (> 100 fmol mg(-1) protein) and eight breast tumours with low PR levels (< 15 fmol mg(-1) protein), as determined by ligand binding assay. The relative expression of D6-PR to wild-type PR mRNA was lower (P< 0.01 ) in normal than in all tumour breast samples. Moreover, a trend to lower (P < 0.1) relative D6-PR expression was observed in high PR tumours, compared to low PR tumours. These data suggest that increased expression of D6-PR occurs during tumorigenesis.

Adult↗

PubMed1999

Variant estrogen receptor-alpha messenger RNA expression in hormone-independent human breast cancer cells.

T5-PRF cells are insensitive to the growth-stimulatory effects of estrogen while still retaining expression of estrogen receptor-alpha (ER-alpha). In the apparent absence of ligand, T5-PRF cells have a 3. 6+/-0.5 (s.e.m.)-fold increased basal ER-alpha activity and elevated basal progesterone receptor levels compared with the parent, estrogen-sensitive, T5 cells. Long-range ER-alpha reverse transcription-PCR was performed to characterize variant ER-alpha mRNA expression in the two cell lines. An increased relative expression of an exon 3/4-deleted ER-alpha mRNA variant was found in T5-PRF. Recombinant expression of this ER-alpha variant resulted in significantly increased estrogen responsiveness, as well as a trend to increased basal ligand-independent activity when expressed with wild-type ER-alpha in ER-negative cell lines, as well as significantly increasing both ligand-independent and estrogen-induced ER-alpha transcriptional activity when expressed in parental T5 cells. These results suggest a role for altered variant ER-alpha in ligand-independent activation of ER-alpha which may contribute to hormone independence in breast tumors.

Breast Neoplasms↗

PubMed1999

The human orphan receptor PXR messenger RNA is expressed in both normal and neoplastic breast tissue.

The expression of PXR mRNA and a variant PXR mRNA, deleted in 111 nucleotides in the ligand-binding domain, was detected by reverse transcription-PCR amplification in both normal and neoplastic human breast tissues. The level of PXR mRNA did not differ between breast tumors and their adjacent matched normal breast tissues. However, the expression of PXR mRNA did vary among breast tumors. A statistically significant inverse relationship was found between the level of PXR mRNA expression and estrogen receptor (ER) status, as defined by ligand binding analysis. The level of PXR mRNA expression in ER+ tumors (median = 22.4, n = 15) was significantly lower (P = 0.04) than the level of PXR mRNA expression in ER-tumors (median = 46.7, n = 15). No relationship with progesterone receptor status was found. These data raise the possibility that PXR has a role in human breast tissues.

Alternative Splicing↗

PubMed1998

Estrogen regulates the association of intermediate filament proteins with nuclear DNA in human breast cancer cells.

In a previous study we showed that the levels of the intermediate filament proteins, cytokeratins 8, 18, and 19, in the nuclear matrix-intermediate filament (NM-IF) fraction from the hormone-dependent and estrogen receptor (ER)-positive human breast cancer cell line T-47D5 were regulated by estrogens. In contrast, estrogens did not regulate the cytokeratins in the NM-IF fraction of the hormone-independent and ER-positive cell line, T5-PRF. In this study, human breast cancer cells were treated with cis-diamminedichloroplatinum to cross-link protein to nuclear DNA in situ, and proteins bound to DNA were isolated. We show that cytokeratins 8, 18, and 19 of T-47D5 and T5-PRF were associated with nuclear DNA in situ. The levels of the cytokeratins 8, 18, and 19 bound to nuclear DNA or associated with the cytoskeleton of T-47D5 human breast cancer cells decreased when estrogens were depleted or the pure antiestrogen ICI 164,384 was added. In contrast, the cytokeratin levels associated with nuclear DNA or cytoskeleton were not significantly affected by estrogen withdrawal or antiestrogen administration in T5-PRF cells. These observations suggest that estrogen regulates the organization of nuclear DNA by rearrangement of the cytokeratin filament network in hormone-dependent, ER-positive human breast cancer cells and that this regulation is lost in hormone-independent, ER-positive breast cancer cells.

Breast Neoplasms↗

PubMed1998

Elevated mitogen-activated protein kinase activity in estrogen-nonresponsive human breast cancer cells.

The mitogen-activated protein kinase (MAPK) signal transduction pathway plays an essential role in cell cycle progression and can be activated by many growth factor/mitogen pathways including estrogen. MAPK has also been implicated in ligand-independent activation of estrogen receptor-alpha (ER-alpha). The development of estrogen-independent growth in breast cancer is likely a first step in progression to hormone independence and antiestrogen resistance. We examined MAPK expression and activity in T5-PRF and T5 human breast cancer cells. T5-PRF is an estrogen-nonresponsive cell line developed from T5 cells by chronically depleting the cells of estrogen in long-term culture. MAPK activity measured in vitro was significantly higher (P < 0.05) in T5-PRF compared with T5 cells. Western blot analyses showed increased levels of active dually phosphorylated MAPK in T5-PRF cell extracts compared with T5. The increased activity and expression of MAPK may contribute to the estrogen nonresponsive growth phenotype and ligand-independent activity of ER in T5-PRF cells.

Breast Neoplasms↗

PubMed1998

Altered estrogen receptor alpha and beta messenger RNA expression during human breast tumorigenesis.

Using a multiplex reverse transcription-PCR assay, we compared the relative expression of estrogen receptor (ER) alpha and ER-beta mRNA between adjacent samples of normal breast tissue and matched primary breast tumors obtained from 18 different patients. Within this cohort, 7 tumors were ER negative, and 11 tumors were ER positive, as determined by the ligand binding assay. No differences in the ratio of ER-alpha:ER-beta expression were observed in the ER-negative cohort. However, in the ER-positive cohort, a significantly (P < 0.02) higher ER-alpha:ER-beta ratio was observed in the tumor compared with that of the normal tissue component. Our data revealed that the increase in the ER-alpha:ER-beta ratio was due primarily to a significant (P < 0.05) increase in ER-alpha mRNA expression in conjunction with a lower ER-beta mRNA expression in the tumor compared with that of the normal compartment in some, but not all, ER-positive cases. These results suggest that the role of ER-alpha- and ER-beta-driven pathways and/or their interaction change during breast tumorigenesis.

Breast↗