Involving AP-2 transcription factor in connexin 26 up-regulation during pregnancy and lactation.

Gap junction connexin 26 (Cx26) is up-regulated in mammary epithelial cells during pregnancy and lactation. To understand the transcriptional regulation of Cx26, we identified a protected DNase I footprint region (-140 to -113) in the rat Cx26 promoter. This rCx26 Promoter Footprinting Region, or CPFR, contains an Sp binding site (CCGCCC) overlapping with an AP-2 binding site (GCCCGCGGC), and is evolutionarily conserved. Nuclear extracts from rat mammary glands and human MCF-10 mammary epithelial cells formed protein-DNA complexes with the labeled CPFR probe in the electrophoretic mobility shift assay (EMSA), and these complexes were markedly enhanced during pregnancy and lactation. Antibody supershift analysis further identified the presence of Sp1, Sp3, and AP-2 in these binding complexes. Human mammary epithelial MCF-10A and MCF-12A cells were transiently transfected with chimeric mutant rCx26 promoter/luciferase reporter constructs, and luciferase activities measured. Mutations along the CPFR fragment drastically reduced the promoter activity, specially at the Sp/AP-2 overlapping site. Cotransfection of AP-2 with rCx26 promoter/reporter constructs into MCF-10 cells markedly induced the reporter activity. These data infer that AP-2, along with previously reported Sp transcription factors, is involved in the up-regulation of Cx26 gene during pregnancy and lactation.

Modulation of the connexin26 tumor suppressor gene expression through methylation in human mammary epithelial cell lines.

BACKGROUND: Normal mammary epithelial cells express mainly gap junction connexin 26 (Cx26) that is either reduced or absent in breast cancers. Since connexin gene mutations are rare we examined if Cx26 gene repression is related to hypermethylation. MATERIALS AND METHODS: Five breast epithelial cell lines were examined for Cx26 mRNA expression and hypermethylation. Treatment with a DNA methyltransferase inhibitor, 5-Aza-2'-deoxycytidine (5-Aza-CdR), was carried out to determine if Cx26 gene expression could be upregulated. RESULTS: Cx26 expression was easily detectable in an immortalized human mammary epithelial cell line (MCF-10) and markedly diminished (MDA-MB231) or undetectable in (MCF-7, BT-20, T47-D) breast cancer cell lines. Hypermethylation of the Cx26 5' region was observed in MCF-10 and MCF-7 cells. Treatment with 5-Aza-CdR resulted in slight or no induction in Cx26 expression in breast cancer cell lines. CONCLUSIONS: Hypermethylation is unlikely to be a major mechanism for Cx26 gene repression in human mammary cancer cell lines.

Differential up-regulation of gap junction connexin 26 gene in mammary and uterine tissues: the role of Sp transcription factors.

The mRNA and protein expressions of connexin 26 (Cx26) in rat mammary gland and uterus can be up-regulated during pregnancy as well as by the administration of human CG (hCG). In the present study, we found that the time course and magnitude of Cx26 induction by hCG was different in these two tissues. The molecular mechanism underscoring this difference was therefore investigated. We had previously demonstrated that both Sp1 and Sp3 transcription factors play a functional role in Cx26 expression. By the electrophoretic mobility shift assay, nuclear extracts from both virgin mammary gland and uterus were capable of binding to a labeled oligonucleotide probe that contained the proximal GC box and formed three protein-DNA complexes (C1, C2, and C3). In the mammary gland, pregnancy enhanced the intensity of all three complexes, whereas in the uterine tissue there was a decrease in the C2 and C3 complexes and an emergence of a new major component, C4 complex. In the supershift study, the C1 complex could be supershifted only by an antibody against Sp1, whereas C2, C3, and C4 could all be supershifted by an antibody against Sp3, suggesting a potential presence of Sp3 isoforms of various sizes. We therefore conclude that the basal Sp profiles in virgin mammary gland and uterine tissue are similar. However, in response to pregnancy, the changes in Sp profile are tissue specific and may account for the temporal and quantitative differences between these two tissues in Cx26 induction.

Mapping and characterization of the basal promoter of the human connexin26 gene.

Connexin26 (Cx26) is a major gap junction protein expressed in mammary and endometrial epithelial cells. Previously, we have cloned the genomic upstream sequence of the human connexin26 gene. In this paper, we studied the structure and function of its basal promoter. Various 5'-flanking regions of the human Cx26 gene were inserted upstream of the bacterial chloramphenicol acetyltransferase (CAT) reporter gene and transfected into human immortalized mammary MCF-10A and MCF-12A cell lines and endometrial RL95-2 cancer cell line. Through CAT reporter gene analysis, we identified the basal promoter of human Cx26 gene in the proximal 5'-flanking region from -128 to +2 (relative to the transcription initiation site). Further deletion analyses suggested that the critical regulatory area was located within a 29 bp region (from -97 to -69), where two GC consensus boxes (CCGCCC) resided, one at -93 and the other at -81. Labeled oligonucleotides encompassing these two GC box DNA sequences could bind the nuclear extracts from MCF-12A and RL95-2 cells in the electrophoretic mobility shift assay. These binding complexes could be competitively reduced by non-labeled self or Sp1 consensus oligonucleotide, and supershifted by antibodies against either Sp1 or Sp3. Mutations in the core sequence of these two GC boxes from CCGCCC to CCGAAC caused a loss of competitive ability and also produced a drastic reduction of basal promoter activity when integrated into promoter/reporter constructs. Furthermore, co-transfection of Sp1 and/or Sp3 expressing plasmids could trans-activate the expression of human Cx26 promoter/reporter constructs in Drosophila Schneider line 2 (SL2) cells. Taken together, these data indicated that the two GC boxes in the proximal promoter region play an important role in the control of human Cx26 gene expression.

Gap junction Cx26 gene modulation by phorbol esters in benign and malignant human mammary cells.

Connexin (Cx) 26, a major gap junction protein expressed in mammary epithelial cells, has been considered to be a tumor suppressor gene candidate. This study investigated the molecular mechanism of transcriptional up-regulation of Cx26 by phorbol ester (TPA) in human immortalized MCF-10 mammary epithelial cells and MDA-MB-231 mammary cancer cells. Such up-regulation was mediated through the protein kinase C pathway and could be blocked by the PKC inhibitor, calphostin C. Based on the results of the nuclear run-on assay, there was a TPA-induced increase in the rate of transcriptional initiation. We identified a TPA-induced DNase I hypersensitivity (DH) region approximately 1 kb 5' upstream of the ATG translation starting site. Sequence analysis revealed that this DH region was located in intron 1 and contained two TRE-like TGAT/ATCA elements, two 5'TTCA3' motifs and a 5'AGGAAG3' PEA3 motif. Both TRE-like elements were capable of binding AP1. TPA inducibility of this DH region was seen by the CAT reporter assay and appeared to be direction-dependent suggesting a functional cooperation between PEA3/TTCA and TRE.

Direct modulation of tumor suppressor connexin 26 gene by human chorionic gonadotropin in rat mammary glands.

Human chorionic gonadotropin (hCG) has been shown to reduce the incidence of carcinogen-induced rat mammary tumors. Because connexin 26 (Cx26), a tumor suppressor gene candidate, can be up-regulated in mammary epithelial cells during lactation, we examined the in vivo and ex vivo effects of hCG on Cx26 expression in rat mammary tissues and used its effect on the expressions of beta-casein and Cx43 as controls. The Cx26 mRNA and protein expressions were up-regulated by daily administrations of 100 units of hCG, starting on day 5 and reaching a 14-fold maximum increment on days 16 through 21. It remained elevated above the basal level even 20 days after hCG withdrawal. The changes in beta-casein expression ran parallel to that of Cx26, whereas the expression of Cx43 was down-regulated. There was no correlation between steroidal hormone levels and Cx26 expression, except for the first 5 days of hCG treatment. In the ex vivo organ culture system, exposure of mammary glands to 10 units/ml hCG for 5 days up-regulated Cx26 but had no effect on beta-casein expression. These results imply a direct induction of the tumor suppressor Cx26 gene by hCG in mammary epithelial cells, a mechanism unrelated to lactation.

Upstream genomic sequence of the human connexin26 gene.

Human connexin 26 (Cx26) has been considered to be a candidate suppressor gene in mammary epithelial cells. To gain insight into the transcriptional regulation of this gene, we have cloned and sequenced the 5' portion of the gene, which extends 4.8 kb upstream from the ATG translation start site. The 3' end of the non-coding exon 1 (160 bp) is located at 3149 bp upstream from the 5' end of exon 2. Comparison between the human Cx26 gene and the mouse gene reveals a highly conserved promoter region with 81% homology. In addition to six GC boxes and two GT boxes, a TTAAAA box is located at -24 to -19 bp upstream of the transcription start point. Analogous to the mouse beta-casein gene, the promoter region of the human Cx26 gene also contains a YY1-like binding site and a consensus mammary gland factor binding site.

Psychological symptoms and disease-free and overall survival in women with stage II breast cancer. Cancer and Leukemia Group B.

BACKGROUND: The possible link between psychological factors and length of cancer survival has generated a literature of contradictory findings. Associations usually have not been found when general psychological symptoms are assessed. Associations usually have been found for predictors related to expressive versus repressive emotional coping (e.g., depression, "fighting spirit," hostility, and type C personality); however, even these associations have been relatively small, when compared with those for medical factors. Yet few studies have adequately controlled for medical and treatment-related factors. PURPOSE: Within a Cancer and Leukemia Group B (CALGB) national clinical trial of four adjuvant therapy regimens for stage II breast cancer (CALGB 8082), this study prospectively examined the contribution of potential psychological predictors to length of disease-free and overall survival over a 15-year period. METHODS: Subjects were 280 women with stage II breast cancer, out of a total of 899, who were randomly assigned to receive CMFVP (cyclophosphamide-methotrexate-fluorouracil-vincristine-prednisone) for two 6-week cycles or six 4-week cycles, then subsequently randomly assigned to receive or not to receive VATH (vinblastine-doxorubicin-thiotepa-fluoxymesterone). Subjects were recruited during the period between October 1980 and August 1984, inclusive, and followed until January 1996. Prior to chemotherapy, psychological symptoms were assessed using the Symptom Check List-90-Revised (SCL-90-R). SCL-90-R scores were trichotomized into categories representing high, medium, and low distress. Basic base-line sociodemographic data (including age, ethnicity, education, and marital status) and medical data (including lymph node status, estrogen receptor status, menopausal status, and performance status) were collected. Subjects with psychosocial data differed from those without psychosocial data solely in their higher percentage of classification in the mild limitation category of the Zubrod (Eastern Cooperative Oncology Group) performance status rating (subjects with psychosocial data: 14%; subjects without psychosocial data: 8%). RESULTS: In stepwise Cox regression analyses that controlled for sociodemographic and medical variables, there was no significant predictive effect of the level of distress (as measured by the SCL-90-R trichotomized scores) on length of disease-free and overall survival of the study subjects. Risk ratios for low versus high distress were 1.01 (95% confidence interval [CI] = 0.62-1.66) for disease-free survival and 1.03 (95% CI = 0.58-1.82) for overall survival. CONCLUSIONS: This study failed to provide evidence that psychological factors contributed to length of disease-free or overall survival of women who received adjuvant chemotherapy (either CMFVP alone or CMFVP followed by VATH) for treatment of stage II breast cancer. IMPLICATIONS: In the context of far more potent medical factors, the contribution of psychological factors to disease-free and overall survival is likely to be relatively small. Future research should focus on specific theory-driven predictors rather than on general psychological symptoms. Moreover, it should be based on clinical studies using a controlled, prospective design, in which the effects of medical factors may be distinguished and psychological predictors are clear antecedents of survival outcomes.

Cyclic AMP modifies the cellular distribution of connexin43 and induces a persistent increase in the junctional permeability of mouse mammary tumor cells.

Direct communication between cells via gap junctions is thought to be an important component of homeostasis and coordinated cellular responses to external signals. We investigated how the second messenger cAMP exerts its effects on junctional communication in a mouse mammary tumor cell line, MMT22. Junctional permeance was quantitatively assessed using dye microinjection and video microscopy. An increase of permeance was found after exposure to 8-bromo-cAMP, being detectable after 30 minutes of treatment and attaining a fourfold higher level of permeance by 24 hours. This elevated level was maintained with continuous exposure to 8-bromo-cAMP for seven days. The permeability change was accompanied by an increase in gap junctions as shown by freeze-fracture electron microscopy and by confocal microscopy using antibodies directed against the gap junction protein, connexin43. The amount of detergent-insoluble connexin43 also increased with 8-bromo-cAMP treatment, and most of the increase could be attributed to an increase of slower migrating (i.e. phosphorylated) species of connexin43. However, connexin43 mRNA and the total cellular content of connexin43 did not change over this period of exposure to 8-bromo-cAMP, as shown by densitometric analyses of northern and western blots. We conclude that 8-bromo-cAMP affects the distribution of connexin43 such that a greater proportion of the protein is utilized for channel formation. Since these changes were relatively slow to develop and persisted with prolonged exposure to 8-bromo-cAMP, it is possible that the junctional permeability of these mammary tumor cells is linked to the 'basal' level of cAMP, i.e. levels maintained by the cells in accordance with a particular cell state.

Analysis of multiple gap junction gene products in the rodent and human mammary gland.

The expression and localization of three different connexins (alpha 1, Cx43; beta 1, Cx32; and beta 2, Cx26) were analyzed in human, mouse, and rat mammary glands by PCR analysis of reverse-transcribed RNA (RT-PCR) and indirect immunohistochemistry. For the rodent mammary gland, the study included different physiological stages of development during nonpregnancy, pregnancy, lactation, and postweaning. RT-PCR amplification revealed a constitutive expression of RNA for alpha 1 connexin in all three species. In contrast, both beta 1 and beta 2 transcripts were expressed only during lactation in the rodent mammary gland. Specifically, immunohistochemistry showed that the expression of all three connexins was restricted to specific cell types, and it varied according to the physiological activity of the organ. In particular, alpha 1 antigen was detected only between myoepithelial cells, the contractile cells surrounding alveoli and ductal systems in the mammary gland, while beta 1 and beta 2 antigens were localized solely at the basolateral border of alveolar secretory cells. The level of beta 1 and beta 2 connexins was increased in the rodent mammary gland during lactation. No staining for either beta connexin was detected in human mammary gland or in that of nonpregnant, pregnant, or postweaning rodents. The inducible coexpression of beta 1 and beta 2 antigens in luminal cells of the lactating rodent suggests a possible role for these connexins in the coordination of the secretory epithelium.

Alternating chemotherapy regimens for patients with metastatic breast cancer. A pilot study based on tumor marker kinetics. Cancer and Leukemia Group B.

BACKGROUND: Chemotherapy is most effective when applied during the biologically active stage of tumor cells. According to the authors' previous tumor marker kinetic study, methotrexate plus 5-fluorouracil (MF) was found to yield either a cytolytic effect in an MF-sensitive tumor cell population or a cytostatic effect in an MF-resistant population. In the latter, the suppressive effect was transient and the biologic activity resumed in one week after MF administration. METHODS: Based on this marker kinetic study, an alternating chemotherapy program was designed to study its antitumor and side effects. Methotrexate (M) (200 mg/m2) and 5-fluorouracil (F) (500 mg/m2) were administered intravenously on day 1 followed 24 hours later by leucovorin (L) (10 mg/m2 orally every 6 hours for 6 doses). Cyclophosphamide (C) 300 (mg/m2), doxorubicin (A) (50 mg/m2), and vincristine (V) (1 mg/m2) were given on day 8. The MFL/CAV was given every 4 weeks. RESULTS: Forty-nine patients with metastatic breast cancer were enrolled; 41 were eligible. There were 5 complete and 23 partial remissions, producing a total response rate of 68%. In 15 patients with liver metastases, the response rate was 73% and the median survival 13.7 months, results superior to those previously reported for this subgroup of patients. Side effects were manageable. CONCLUSIONS: This regimen, which can be given safely in an outpatient setting, yielded encouraging response and survival rates in patients with visceral-dominant disease with poor prognoses.

Measurement of gap junctional communication by fluorescence activated cell sorting.

Cell-to-cell communication via gap junctions has played a fundamental role in the orderly development of multicellular organisms. Current methods for measuring this function apply mostly to homotypic cell populations. The newly introduced Fluorescence Activated Cell Sorting (FACS) method, albeit with some limitations, is simple, reliable, and quantitative in measuring the dye transfer via gap junctions in both homotypic and heterotypic cell populations. In the homotypic setting, the result in dye transfer from the FACS method is comparable to the scrape-loading and microinjection methods. Using this FACS method, we observed a decline of cell-to-cell communication in transformed and cancer cells. We also observed a differential degree of communication between two heterotypic cell populations depending on the direction of dye transfer.

Regulation of insulin-like growth factors by antiestrogen.

Insulin-like growth factors are potent mitogens for breast cancer cell proliferation. This effect is modulated by the circulatory and extracellular IGFBPs as well as by the affinity of ligand binding receptors on the target cells. Antiestrogens have been shown to reduce both circulatory and microenvironmental IGF levels and thus suppress the IGF-I-induced growth of both ER-positive and ER-negative breast cancer cells. However, the effects of antiestrogens in down regulation of type I IGF receptor and in altering the autophosphorylation tyrosine kinase activity of EGF receptors are mainly observed in ER-positive cells. Furthermore, alteration of IGFBP by antiestrogens such as a marked increase of IGFBP-I production have been shown to inhibit the proliferative effect of IGF-I on ER-positive, but stimulate this effect, on ER-negative cells. Such differential effects from IGF receptor and IGFBP may explain the clinical outcome that tumor regression from antiestrogens is mainly observed in ER-positive type. This assumption based on IGF regulation alone is certainly an oversimplistic view amid the complexity of autocrine, paracrine, and endocrine functions.

Stability of HER-2/neu expression over time and at multiple metastatic sites.

BACKGROUND: Amplification and over-expression of the HER-2/neu oncogene (also known as c-erbB-2) occurs in 20%-30% of invasive breast carcinomas. The extent to which HER-2/neu expression changes over time in association with tumor progression or is heterogeneous at different metastatic sites has received only limited study. PURPOSE: Our purpose was to determine whether primary tumors differ from metastases in HER-2/neu protein content or whether metastases are heterogeneous in regard to HER-2/neu expression. METHODS: In a retrospective study, we examined tumor tissue obtained at autopsy from two to five metastatic organ sites in each of 30 patients who died with metastatic breast carcinoma. Using an immunoperoxidase technique, we stained archival formalin-fixed, paraffin-embedded tissue sections with a monoclonal antibody to the 185-kilodalton protein product (p185) of the HER-2/neu gene. RESULTS: The tissue from eight of 30 patients showed strong diffuse reactivity for p185 at all metastatic sites examined. Tissues from six patients showed faint staining and tissues from 15 were negative, again with a congruent staining pattern. A single case showed discordant staining, in that two of four metastases showed faint staining, whereas the other two showed strong immunoreactivity. In 14 cases, we were able to obtain paraffin blocks from the original biopsy or surgical resection of the primary breast lesion. For these 14 patients, the average length of time between initial diagnosis and death was 4 years (range, 2-9). There was good correlation between results from autopsy and original surgical tissues. CONCLUSIONS: Expression of HER-2/neu appears to be relatively stable over time and is generally congruent at different metastatic sites. IMPLICATIONS: The fact that p185 immunoreactivity is rarely heterogeneous is encouraging, both for the potential use of HER-2/neu-related proteins as serum tumor markers and for innovative therapies targeted at p185 expression.

Detection of c-erbB-2 activation in paraffin-embedded tissue by immunohistochemistry.

Commercially available monoclonal antibodies were tested for their ability to detect increased levels of c-erbB-2 protein in formalin-fixed, paraffin-embedded breast carcinomas. Of five antibodies studied, four (TAB-250, CB11, 3B5, and N3/D10) showed strong cytoplasmic membrane reactivity in 23% (11 of 47) of routinely processed tumors, although interpretation of the immunoreactivity with 3B5 and N3/D10 occasionally was difficult due to cytoplasmic granular staining. Since the c-erbB-2 oncogene is activated by DNA amplification and overexpression of mRNA and protein, the same tumors were analyzed for c-erbB-2 activation by other techniques. c-erbB-2 activation in these 11 tumors was confirmed by immunohistochemistry of frozen tissue (nine of nine tumors), in situ hybridization (nine of 11 tumors), and Southern blot analysis (five of eight tumors). In some of these tumors the failure to demonstrate c-erbB-2 DNA amplification may be due to the small percentage of malignant cells. One additional tumor showed probable c-erbB-2 protein overproduction based on strong immunoreactivity with two antibodies (TAB-250 and CB11), although no definite activation could be demonstrated by additional techniques. Three other tumors (6%) showed equivocal c-erbB-2 protein overproduction based on weak immunoreactivity only with TAB-250, although unequivocal activation could not be demonstrated by additional techniques. The 32 carcinomas (68%) that showed no significant immunoreactivity with any antibodies in routinely processed tissue also showed no detectable c-erbB-2 activation by additional techniques. We conclude that TAB-250 and CB11 are reliable antibodies for detecting c-erbB-2 protein overproduction in routinely processed tissue. TAB-250 also weakly stains a few tumors showing no definite c-erbB-2 activation by other techniques. Two additional antibodies (3B5 and N3/D10) detect c-erbB-2 protein overproduction in paraffin-embedded tissue, but are more difficult to interpret. A fifth antibody, TA-1, is an excellent reagent for use on frozen tissue, but prolonged formalin fixation may impair recognition of its antigenic epitope.

Variations in amplification and expression of the ornithine decarboxylase gene in human breast cancer cells.

The polyamine biosynthetic pathway plays a critical role in the growth of human breast cancer cells. Ornithine decarboxylase (ODC) is a key enzyme in polyamine biosynthesis. To understand the regulation of ODC activity and polyamine accumulation in breast cancer cells, we studied amplification and expression of the ODC gene in four breast cancer cell lines. ODC gene dosage was analyzed by Southern blot hybridization and was 4- to 12-fold higher in T-47D, MDA-MB-231, and BT-20 cell lines than in the MCF-7 cell line. ODC mRNA level was 2- to 3-fold higher in BT-20 and MDA-MB-231 cell lines than in the other two lines. We also measured ODC activity and polyamine concentration in these cell lines, and determined their sensitivity to an ODC inhibitor, difluoromethylornithine (DFMO). BT-20 cells showed significantly higher ODC activity and polyamine concentrations than the other three cell lines. BT-20 cells were resistant to the growth inhibitory effect of DFMO even at 4 mM concentration, whereas the proliferation of MCF-7, T47D, and MDA-MB-231 cells was inhibited by this drug. These results suggest that different transcriptional and post-transcriptional mechanisms control the regulation of ODC gene expression in breast cancer cell lines.