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The effects of the TNF- α blockers infliximab or etanercept on the levels of TNF- α , TNF-receptor 1 (TNF-R1), and TNF-receptor 2 (TNF-R2), as well as the levels of the inflammation markers CRP and IL-6, were measured in ankylosing spondylitis (AS) and rheumatoid arthritis (RA) patients receiving treatment with either compound. We found that RA patients tend to have higher levels of TNF- α than both healthy individuals and AS patients prior to treatment (P < 0.05). We measured greatly increased levels of TNF- α in both the AS and RA etanercept patient groups during the course of treatment, while in the infliximab treated patients, the amount of TNF- α measured remained unchanged. Elevated TNF- α in the etanercept treated patients does not appear to be a significant risk factor for the spontaneous development of further autoimmune diseases in our study group. Increased levels of TNF-R1 were determined in both AS (P < 0.05) and RA (P < 0.001) patients when compared to healthy controls. In AS patients, the levels of TNF-R1 dropped significantly when treated with either infliximab (P < 0.01) or etanercept (P < 0.001). In contrast, the levels of this receptor remained unchanged in RA patients treated with either compound.

In a comparative proteome analysis of peripheral blood mononuclear cells (PBMCs), we analyzed 130 two-dimensional gels obtained from 33 healthy control individuals and 32 patients diagnosed with rheumatoid arthritis (RA). We found 16 protein spots that are deregulated in patients with RA and, using peptide mass fingerprinting and Western blot analyses, identified these spots as belonging to 9 distinct proteins. A hierarchical clustering procedure organizes the study subjects into two main clusters based on the expression of these 16 protein spots, one that contains mostly healthy control individuals and the other mostly RA patients. The majority of the proteins differentially expressed in RA patients when compared with healthy controls can be detected as protein fragments in PBMCs obtained from RA patients. This set of deregulated proteins includes several factors that have been shown to be autoantigens in autoimmune diseases.

The aim of this study was to determine the activation level of the pro-inflammatory transcription factor nuclear factor kappaB (NF-kappaB) in lymphocytes of patients with rheumatoid arthritis (RA) before and during an anti-tumor necrosis factor alpha (TNFalpha) therapy (adalimumab). In addition, we analyzed the inflammatory markers, interleukin 6 (IL-6), and C-reactive protein (CRP) and investigated the expression of rheumatoid factor (RF) and anti-cyclic citrullinated peptide (anti-CCP) autoantibodies in patients' sera. Twenty RA patients and 20 control subjects were investigated. RA patients' characteristics were evaluated by radiography and disease activity score 28 (DAS 28). Twelve weeks of adalimumab therapy was effective in the treatment of RA patients, as shown by a significant improvement of the DAS 28. The inflammatory markers IL-6 and CRP were significantly different in sera of RA patients compared to the control group before the onset of therapy and exhibited a tendency to return to normal levels during the first 12 weeks of therapy. We measured a comparable activation level of NF-kappaB in lymphocytes of control subjects and of RA patients before starting adalimumab therapy. During the following 12 weeks, no significant changes in the activation levels of both NF-kappaB subunits were detected. Serum concentration of RF was significantly lower after 12 weeks, whereas anti-CCP antibody level remained constant.

The aim of this study was to analyse patients with ankylosing spondylitis (AS) during the course of infliximab therapy. The molecular effects were evaluated using lymphocytes and sera that were isolated before therapy began, then again after 2 and 12 weeks from 17 AS patients and compared to those of 24 healthy control individuals. All 17 AS patients responded to treatment with infliximab as assessed using BASDAI. Elevated serum levels of IL-6, CRP and cortisol were reduced to normal levels by the 12 weeks time point. The level of DNA-binding p65 was decreased during the course of infliximab therapy whereas the level of DNA-binding p50 remained elevated until the 12 weeks time point. Taken together, Infliximab is an effective treatment for AS and results in decreased levels of the inflammation markers IL-6 and CRP, and of endogenous cortisol concentration. Unequal alterations in the levels of activated NF-kappaB subunits p50 and p65 might provide insights into the mechanisms of NF-kappaB action and anti-TNF-alpha therapy in AS.

We compared the expression levels of proteins in peripheral blood mononuclear cells (PBMCs) of healthy control individuals to those of patients diagnosed with rheumatoid arthritis (RA) using a proteomics approach. Using two-dimensional electrophoresis we identified 18 proteins that were 2-fold or more highly expressed in patients than in controls, and 11 proteins that were 2-fold or more highly expressed in controls than in patients. Some of these differentially expressed proteins, identified by MALDI-TOF spectrometry, have previously been shown to play a potential role in the pathogenesis of RA. Hierarchical cluster analyses of the data segregated the samples into two groups, one which contained only controls and the other which contained only patients, and was used to compare the expression pattern of these 29 proteins in individual samples with the median expression pattern determined in the healthy control and in the RA patient groups. This analyses was able to predict whether a sample was derived from a rheumatoid arthritis patient or from a healthy individual, suggesting that a comparison of such protein expression patterns may be of diagnostic value.

The human androgen receptor (AR) is a member of the nuclear hormone receptor superfamily. However, in contrast to other members of this family the amino-(N)-terminus of AR harbors the major transactivation function. Previously we have shown that hormone antagonists that bind to the carboxy-terminal ligand-binding domain repress AR through recruitment of corepressors that are recruited to the receptor N-terminus. Here we show by a modified mammalian two-hybrid system that both the AR interacting domains of the coactivator SRC1 and of the corepressor SMRT compete for interaction with the AR N-terminus. In contrast to other members of the nuclear receptor superfamily the LXXLL motifs of SRC1e are not required for this interaction, instead a stretch of 135 amino acids of the glutamine rich region (Qr) of SRC1e is essential to bind to the AR N-terminus. We show that the Qr-region of SRC1 is able to inhibit the interaction of SMRT with AR. Also, we demonstrate that the corepressor mediated repression decreases the antagonist-induced transactivation while, surprisingly, it increases the agonist-induced transactivation. This may indicate that coactivators and corepressors act in concert to dictate the overall receptor-mediated action dependent on the type of ligand.

Objective: To study a possible relationship between expression of the transcription factor glucocorticoid receptor (GR), which mediates antiinflammatory effects, and the transcription factor p50, which mediates proinflammatory effects, in peripheral blood mononuclear cells (PBMC) of patients with rheumatoid arthritis (RA)., Methods: Expression analysis of GR and nuclear factor-kB subunit p50 in PBMC was performed by semiquantitative immunoblotting., Results: GR and p50 expression in PBMC were significantly increased in patients with RA who had never received corticosteroids. In contrast, GR density is decreased in glucocorticoid treated RA patients. In addition, a dependency between increased GR expression and increased p50 expression was found., Conclusion: The pathogenesis of RA is not reflected in diminished GR expression but rather in an increased expression level of GR, as well as increased p50 expression in PBMC. Corticosteroids as the major therapeutic drugs result in a reduction of these increased GR and p50 expression levels.

We describe here an unusual phenomenon in the isolation of protein complexes from eukaryotic cells using expressed GST-fusion proteins. Protein complexes are involved in a large number of regulatory mechanisms. Therefore, the use of tagged fusion proteins is an important tool for isolation of such protein complexes. For this purpose, we used the nuclear factor Alien, described as a corepressor for the thyroid hormone receptor, fused to the eukaryotic eGST and expressed this fusion in human cells. After affinity purification over glutathione-Sepharose using stringent washing steps, we observed several co-purifying bands migrating at molecular weights higher than the GST-Alien fusion protein. These bands appeared specifically in the GST-Alien transfected cell preparations. Surprisingly, using both Western blotting and MALDI-analyses, we revealed that these bands are composed of the GST-Alien protein itself. We hypothesize that overexpressed factors may generate unexpected cross-linking products which can confound the analyses of such affinity-purified complexes. The cross-linking products could not be eliminated by using beta-mercaptoethanol in the gel system and by boiling in SDS-sample buffer. Also, we demonstrate that Western blotting analyses using antibodies directed against both the tag-epitope and the expressed protein of interest can rapidly, reliably, and in a cost-saving manner identify such artifacts, eliminating them from the analyses of potentially interesting interaction partners. Our findings clearly show that the overexpression and purification of proteins from eukaryotic cells may generate unusual structural features that strongly influence complex formation and the migration in SDS-PAGE.

The glucocorticoid receptor (GR) is a ligand-inducible transcription factor which controls the expression of several genes. Its cognate ligand, the glucocorticoids, induces receptor activation by binding to the cytoplasmic located receptor, ultimately leading to translocation of the receptor/hormone complex into the nucleus and the regulation of gene activity. Because glucocorticoids are widely used for suppression of inflammation in rheumatoid arthritis (RA), we investigated whether the expression level of GR is correlated with RA. We designed a study to detect the total amount of GR in lymphocytes of untreated RA patients, glucocorticoid-treated RA patients, and healthy controls. We observed a significant change in the expression levels of GR. Untreated RA patients exhibited a significantly higher amount of GR than the healthy controls, whereas glucocorticoid-treated RA patients showed a strongly decreased receptor density. These results seem to reflect a functional dysregulation of the HPA axis and may lead to a better understanding of the pathogenesis of RA.

Antiandrogens inhibit the ligand-induced transactivation by the androgen receptor (AR) and have a widespread use in the treatment of prostate cancer but their mode of action is not fully understood. Here we show that the ability of the antiandrogen cyproterone acetate (CPA) to inhibit transactivation by the human AR (hAR) involves the corepressor SMRT (silencing mediator for retinoic acid and thyroid hormone receptor). We detect binding of SMRT to hAR when treating with the antiandrogen CPA, but not with the antihormones casodex or hydroxyflutamide. Interestingly, we find that SMRT binds to the N terminus of the hAR. Thereby, SMRT modulates the activity of hAR in receptor-negative CV1 cells. In addition, we have used receptor point mutants that exhibit normal transactivation potential and unchanged partial agonistic activity when treated with CPA, but lack both SMRT binding and SMRT-mediated inhibition of CPA-bound AR. This indicates that mechanisms involved in hAR-mediated transactivation are distinct from antihormone-induced receptor inactivation. Furthermore, we show that treatment of transfected cells with a cAMP analog or coexpression of the catalytic subunit of PKA, known to activate hAR, inhibits the binding of SMRT to the AR. This suggests that the association of SMRT with hAR is regulated at the level of cross-talk mechanisms and that ligand-independent receptor activation is due to corepressor dissociation. Taken together, we provide novel insights in AR regulation, antihormone action, and functional nuclear receptor-corepressor interaction.

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.

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., (Copyright 2000 John Wiley & Sons, Ltd.)

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.

Using semiquantitative reverse transcription-polymerase chain reaction assays, we investigated the expression of variant messenger RNAs relative to wild-type estrogen receptor (ER)-alpha messenger RNA in normal breast tissues and their adjacent matched breast tumor tissues. Higher ER variant truncated after sequences encoding exon 2 of the wild-type ER-alpha (ERC-4) messenger RNA and a lower exon 3 deleted er-alpha variant (ERD3) messenger RNA relative expression in the tumor compartment were observed in the ER-positive/PR-positive and the ER-positive subsets, respectively. A significantly higher relative expression of exon 5 deleted ER-alpha variant (ERD5) messenger RNA was observed in tumor components overall. These data demonstrate that changes in the relative expression of ER-alpha variant messenger RNAs occur between adjacent normal and neoplastic breast tissues. We suggest that these changes might be involved in the mechanisms that underlie breast carcinogenesis.

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.

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., (Copyright 1999 John Wiley & Sons, Ltd.)

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.

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.

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.

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.

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.

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.

Lumican mRNA has been identified as being differentially expressed between different regions of the same human breast tumor. In situ hybridization study of 26 independent breast tumors confirmed the presence of lumican mRNA in fibroblast-like cells within stroma and showed a significant increase of its expression in tumor compared to adjacent normal stroma (P < 0.001). Higher lumican expression was associated with higher tumor grade, lower estrogen receptor levels in the tumor, and younger age of the patients (P < 0.05). Reverse transcription-PCR analysis of total RNA extracted from 19 independent breast tissues exhibiting lesions that are thought to parallel tumor progression also suggests that this proteoglycan is differentially expressed during tumor progression.

Estrogen receptor (ER)-beta mRNA splice variants have been identified in human breast tumors as well as normal human and mouse ovarian, uterine and mammary tissues. In both species transcripts deleted in exons 5 or 6, or 5 + 6 have been characterized by RT-PCR followed by cloning and sequencing. In mouse tissues an ER-beta transcript containing 54 nucleotides inserted in frame between exons 5 and 6 was identified. Interestingly, no equivalent of the mouse inserted transcript was detected in any of the four human tissues analyzed.

The expression of a recently described novel estrogen receptor, ER-beta, was detected in several human breast tumor biopsy samples and several human breast epithelial cell lines using reverse transcription and polymerase chain reaction (RT-PCR) analysis. Cloning and sequencing of the PCR product from a breast tumor confirmed the identity of the sequence with that of the ER-beta mRNA previously reported in human testis. The expression of ER-beta was not correlated with that of ER-alpha, and both ER-alpha positive and ER-alpha negative cell lines expressed ER-beta mRNA. However, some breast tumors and some cell lines coexpress ER-beta and ER-alpha mRNA. Our data support a possible role for ER-beta in human breast cancer.

Several oestrogen receptor variant and mutated mRNA species have been identified in human breast samples and cell lines. Over-expression and altered expression of some of these mRNAs have been correlated with breast tumourigenesis and progression. The following review focuses on the current knowledge available in the scientific literature with respect to the type and characteristics of oestrogen receptor variants and mutations that have been identified as occurring naturally in human breast tissues and cell lines.

The tissue matrix consists of linkages and interactions of the nuclear matrix, cytoskeleton, and extracellular matrix. This system is a dynamic structural component of the cell that organizes and processes structural and functional information to maintain and coordinate cell function and gene expression. We have studied estrogen regulation of nuclear matrix associated proteins, including the intimately connected cytoskeletal intermediate filaments, in T-47D5 human breast cancer cells. Three proteins (identified as cytokeratins 8, 18, and 19) present in the nuclear matrix-intermediate filament fraction (NM-IF) of cells grown in estrogen-replete conditions were dramatically reduced when the cells were grown in acute (1 week) estrogen-depleted conditions. Replacing estrogen in the medium of acute estrogen-depleted cells restored expression of these proteins. T-47D5 cells that are chronically depleted of estrogen (T5-PRF) are estrogen-nonresponsive in culture. These cells overexpressed these three proteins, compared to parent cells grown in the presence of estrogen. Treatment of the T5-PRF cells with estrogen did not lead to further up-regulation of these proteins. Treating T-47D5 cells in estrogen-replete conditions with the antiestrogens 4-hydroxytamoxifen and ICI 164 384 (100 nM, 3 days) resulted in a significant reduction in these proteins, while no effect was seen in long-term chronic estrogen-depleted T-47D5 cells. In conclusion, we have identified NM-IF proteins (cytokeratins 8, 18, and 19) in human breast cancer cells that are estrogen regulated and may play a role in estrogen action in human breast cancer cells.