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Purpose: In preclinical models, established molecular determinants of cellular sensitivity to cyclophosphamide, long a mainstay of chemotherapeutic regimens used to treat breast cancers, include the aldehyde dehydrogenases that catalyze the detoxification of this agent, namely, ALDH1A1 and ALDH3A1. As judged by bulk quantification of relevant catalytic activities, as well as of relevant proteins (ELISAs), tissue levels of these enzymes vary widely in primary and metastatic breast malignancies. Thus, interindividual variation in the activity of either of these enzymes in breast cancers could contribute to the wide variation in clinical responses obtained when such regimens are used to treat these malignancies. Direct evidence for this notion was sought in the present investigation. Methods: Cellular levels of ALDH1A1 and ALDH3A1 in 171 repository human breast tumor (122 primary and 49 metastatic) samples were semiquantified using immunocytochemical staining. Clinical responses were retrieved from the archived medical records of each of 48 metastatic breast cancer sample donors, 26 of whom had been treated with a cyclophosphamide-based chemotherapeutic regimen subsequent to tumor sampling and 22 of whom had not. The premise that cellular levels of ALDH1A1 and/or ALDH3A1 predict clinical responses to cyclophosphamide-based chemotherapeutic regimens was submitted to statistical analysis. Results: Confirming an earlier report, ALDH1A1 and ALDH3A1 levels varied widely in both primary and metastatic breast tumor cells. When measurably present, each of the enzymes appeared to be evenly distributed throughout a given tumor cell population. Retrospective analysis indicated that cellular levels of ALDH1A1, but not those of ALDH3A1, were (1) significantly higher in metastatic tumor cells that had survived exposure to cyclophosphamide than in those that had not been exposed to this drug, and (2) significantly higher in metastatic tumors that did not respond (tumor size did not decrease or even increased) to subsequent treatment with cyclophosphamide-based chemotherapeutic regimens than in those that did respond (tumor size decreased) to such regimens. The therapeutic outcome of cyclophosphamide-based chemotherapy corresponded to cellular ALDH1A1 levels in 77% of cases. The frequencies of false-positives (cyclophosphamide-based chemotherapy not effective when a low level of ALDH1A1 predicted it would be) and false-negatives (cyclophosphamide-based chemotherapy effective when a high level of ALDH1A1 predicted it would not be) were 0.00 and 0.43, respectively. Thus, partial or complete responses to cyclophosphamide-based chemotherapy occurred 2.3 times more often when the ALDH1A1 level was low than when it was high. Conclusions: Given (1) the wide range of ALDH1A1 levels observed in malignant breast tissues, (2) that ALDH1A1 levels in primary breast tumor tissue, as well as those in normal breast tissue, directly reflect ALDH1A1 levels in metastatic breast tumor cells derived therefrom, and (3) the findings reported here, measurement of ALDH1A1 levels in primary breast malignancies and/or normal breast tissue prior to the initiation of chemotherapy is likely to be of value in predicting the therapeutic potential, or lack of potential, of cyclophosphamide and other oxazaphosphorines, e.g. ifosfamide, in the treatment of primary, as well as metastatic, breast cancer, thus providing a rational basis for the design of individualized therapeutic regimens for this disease. Failure to observe the expected inverse relationship between clinical responses to cyclophosphamide-based chemotherapeutic regimens and ALDH3A1 levels was probably because even the highest breast tumor tissue ALDH3A1 level thus far reported appears to be below the threshold level at which ALDH3A1-catalyzed detoxification of oxazaphosphorines becomes pharmacologically meaningful. However, ALDH3A1 levels in certain other malignancies, e.g. those of the alimentary tract and lung, may be of a sufficient magnitude in that regard.

Gap junction connexin 26 lCx26r is uphregulated in mammary epithelial cells during pregnancy and lactation. To understand the transcriptional regulation of Cx26, we identified a protected DNase I footprint region l−140 to −113r in the rat Cx26 promoter. This rCx26 Promoter Footprinting Region, or CPFR, contains an Sp binding site lCCGCCCr overlapping with an APh2 binding site lGCCCGCGGCr, and is evolutionarily conserved. Nuclear extracts from rat mammary glands and human MCFh10 mammary epithelial cells formed proteinhDNA complexes with the labeled CPFR probe in the electrophoretic mobility shift assay lEMSAr, and these complexes were markedly enhanced during pregnancy and lactation. Antibody supershift analysis further identified the presence of Sp1, Sp3, and APh2 in these binding complexes. Human mammary epithelial MCFh10A and MCFh12A cells were transiently transfected with chimeric mutant rCx26 promotersluciferase reporter constructs, and luciferase activities measured. Mutations along the CPFR fragment drastically reduced the promoter activity, specially at the SpsAPh2 overlapping site. Cotransfection of APh2 with rCx26 promotersreporter constructs into MCFh10 cells markedly induced the reporter activity. These data infer that APh2, along with previously reported Sp transcription factors, is involved in the uphregulation of Cx26 gene during pregnancy and lactation. Mol. Reprod. Dev. 59:17–24, 2001. © 2001 WileyhLiss, Inc.

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.

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-pred -nisone) 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.

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.

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/ m 2 ) and 5-fluorouracil (F) (500 mg/m 2 ) were administered intravenously on day 1 followed 24 hours later by leucovorin (L) (10 mg/m 2 orally every 6 hours for 6 doses). Cyclophosphamide (C) 300 (mg/m 2 ), doxorubicin (A) (50 mg/ m 2 ), and vincristine (V) (1 mg/m 2 ) 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

Controversy exists in using carcinoembryonic antigen (CEA) for monitoring the clinical course of breast cancer. In this study, the kinetics of two plasma tumor markers, CEA and CA15-3, immediately after the initiation of chemotherapy were assessed in 30 patients with advanced breast cancer. Four distinct kinetic patterns were seen. Two patterns fitted the expected relationship where the plasma marker increased during tumor progression (nine patients), and declined in tumor regression (five patients). The third pattern was paradoxical in that objective tumor regression in eight patients was associated with an acute surge of these markers followed by a steady decline. The doubling times for both CEA and CA15-3 were immediately shortened four-fold after therapy suggesting tumor cytolysis in treatment responders. Equally paradoxical was the fourth pattern where tumor progression in eight patients was associated with a rapid and transient decline of markers followed by rebounds. Such a rapid decline may be due to a suppression of marker release, as demonstrated in an in vitro study. Adequate knowledge of these putative paradoxical patterns will permit their effective use in monitoring the disease course and perhaps in the early prediction of the therapeutic response.

The type I insulin-like growth factor receptor (IGF1R) and insulin receptor (IR) are structurally and functionally related heterotetrameric receptors. Activation of IGF1R has been shown to regulate breast cancer cell biology, and it has become an attractive therapeutic target. Most strategies have focused on targeting IGF1R alone without affecting IR levels given the known physiologic functions of IR. Human breast cancer cell lines and tissues revealed mRNA expression of both IGF1R and IR. Because αβ chains of IGF1R and IR form hybrid receptors, we hypothesized that agents solely targeting IGF1R may affect tumor biology mediated by IGF1R/IR hybrids and IR. We used small interfering RNA (siRNA) technology to specifically down-regulate IGF1R by 60% to 80% in the MDA-435/LCC6 cell line, which was sufficient to diminish activation of IGF1R by IGF-I. IGF1R down-regulation by siRNA did not affect IR levels but, interestingly, sensitized cells to insulin activation of downstream signaling pathways in several breast cancer cell lines. IGF1R siRNA treatment diminished hybrid receptor formation, suggesting that specific down-regulation of IGF1R resulted in enhanced holo-IR formation. In addition, IGF1R down-regulation increased insulin binding consistent with the formation of an increased number of holo-IR on the cell surface. Accordingly, insulin-stimulated glucose uptake was enhanced on IGF1R down-regulation. In conclusion, our data suggest that specific siRNA targeting of IGF1R alone in breast cancer increases insulin sensitivity. Because IR also activates signaling pathways similar to IGF1R in breast cancer cells, agents targeting both receptors may be necessary to disrupt the malignant phenotype regulated by this growth factor system. [Cancer Res 2007;67(1):391–7]

In preclinical models, established molecular determinants of cellular sensitivity to cyclophosphamide, long a mainstay of chemotherapeutic regimens used to treat breast cancers, include the aldehyde dehydrogenases that catalyze the detoxification of this agent, namely, ALDH1A1 and ALDH3A1. As judged by bulk quantification of relevant catalytic activities, as well as of relevant proteins (ELISAs), tissue levels of these enzymes vary widely in primary and metastatic breast malignancies. Thus, interindividual variation in the activity of either of these enzymes in breast cancers could contribute to the wide variation in clinical responses obtained when such regimens are used to treat these malignancies. Direct evidence for this notion was sought in the present investigation.Cellular levels of ALDH1A1 and ALDH3A1 in 171 repository human breast tumor (122 primary and 49 metastatic) samples were semiquantified using immunocytochemical staining. Clinical responses were retrieved from the archived medical records of each of 48 metastatic breast cancer sample donors, 26 of whom had been treated with a cyclophosphamide-based chemotherapeutic regimen subsequent to tumor sampling and 22 of whom had not. The premise that cellular levels of ALDH1A1 and/or ALDH3A1 predict clinical responses to cyclophosphamide-based chemotherapeutic regimens was submitted to statistical analysis.Confirming an earlier report, ALDH1A1 and ALDH3A1 levels varied widely in both primary and metastatic breast tumor cells. When measurably present, each of the enzymes appeared to be evenly distributed throughout a given tumor cell population. Retrospective analysis indicated that cellular levels of ALDH1A1, but not those of ALDH3A1, were (1) significantly higher in metastatic tumor cells that had survived exposure to cyclophosphamide than in those that had not been exposed to this drug, and (2) significantly higher in metastatic tumors that did not respond (tumor size did not decrease or even increased) to subsequent treatment with cyclophosphamide-based chemotherapeutic regimens than in those that did respond (tumor size decreased) to such regimens. The therapeutic outcome of cyclophosphamide-based chemotherapy corresponded to cellular ALDH1A1 levels in 77% of cases. The frequencies of false-positives (cyclophosphamide-based chemotherapy not effective when a low level of ALDH1A1 predicted it would be) and false-negatives (cyclophosphamide-based chemotherapy effective when a high level of ALDH1A1 predicted it would not be) were 0.00 and 0.43, respectively. Thus, partial or complete responses to cyclophosphamide-based chemotherapy occurred 2.3 times more often when the ALDH1A1 level was low than when it was high.Given (1) the wide range of ALDH1A1 levels observed in malignant breast tissues, (2) that ALDH1A1 levels in primary breast tumor tissue, as well as those in normal breast tissue, directly reflect ALDH1A1 levels in metastatic breast tumor cells derived therefrom, and (3) the findings reported here, measurement of ALDH1A1 levels in primary breast malignancies and/or normal breast tissue prior to the initiation of chemotherapy is likely to be of value in predicting the therapeutic potential, or lack of potential, of cyclophosphamide and other oxazaphosphorines, e.g. ifosfamide, in the treatment of primary, as well as metastatic, breast cancer, thus providing a rational basis for the design of individualized therapeutic regimens for this disease. Failure to observe the expected inverse relationship between clinical responses to cyclophosphamide-based chemotherapeutic regimens and ALDH3A1 levels was probably because even the highest breast tumor tissue ALDH3A1 level thus far reported appears to be below the threshold level at which ALDH3A1-catalyzed detoxification of oxazaphosphorines becomes pharmacologically meaningful. However, ALDH3A1 levels in certain other malignancies, e.g. those of the alimentary tract and lung, may be of a sufficient magnitude in that regard.

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.

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.

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 β-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.

Systemic treatments with hormones and/or chemotherapy attained outstanding success in producing objective regressions of local and distant metastases of breast cancer. With these demonstrations, similar therapies were employed in the adjuvant setting for stage II patients resulting in improved disease-free and overall survival (1,2). For locally advanced disease, systemic treatments caused substantial shrinkage of unresectable primary tumors (3,4). Because of the poor prognosis of stage III breast cancer, a positive approach to its management has been neglected or at least variable. This has been associated, in part, with the changing of staging systems of breast cancer. Further confusion occurred with clinical trials that incorporated stages IIB and III breast cancer. For clarification in this report, reference will be made to stage IIIB breast cancer (5,6). Stage IIIB breast cancer consists of tumors of any size with direct extension to the chest wall or skin (T4) with axillary node involvement (N1 and ...

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.

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.

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.

Reversal of myelofibrosis and splenomegaly is described in a 41 year old woman with metastatic breast cancer. After intensive chemotherapy and hormonal therapy, the tumor regressed, the splenomegaly receded, the hemogram showed no abnormalities, and the dense collagen and reticulin fibers in the marrow disappeared. The severe thrombocytopenia and leukoerythroblastosis noted before therapy were not obstacles to clinical management. In our report we document that myelofibrosis associated with breast cancer is not an ominous sign. Patients may benefit from an intensive, but well titrated, therapeutic program.

Previous reports have shown binding of peanut agglutinin (PNA) by immunoperoxidase techniques in normal, benign proliferative, and malignant breast epithelia. Correlation of binding with maturity, secretory activity, hormonal milieu, and tissue hormone receptor content has been described. To investigate the relationship between PNA staining by the avidin-biotin complex technique and estrogen receptor (ER) content, 79 breast tumors of different types and known tissue ER content were studied. Thirty-eight of 50 ER-positive cases were PNA-positive. Twenty of 29 ER-negative cases were PNA-positive. Statistical analysis shows the two factors to be independent (0.5 less than P less than 0.9). The literature on blood group antigens in breast carcinoma and histochemical applications of PNA is reviewed.

We studied the relative efficacy of polyamines to facilitate the binding of estrogen receptor to poly(dA-dC).poly(dG-dT). In the absence of polyamines, 1,400 micrograms/ml of this polynucleotide eluted 50% of bound estrogen receptor from DNA-cellulose. In contrast, 50% estrogen receptor was eluted by 65 micrograms/ml of poly(dA-dC).poly(dG-dT) complexed with 150 microM spermidine. Putrescine and spermine also enhanced the ability of poly(dA-dC).poly(dG-dT) to elute estrogen receptor, but the magnitude of the effect was not as high as that of spermidine. Control experiments with calf thymus DNA and poly(dA-dT).poly(dA-dT) showed 6- and 3-fold increase, respectively in their affinity for estrogen receptor in the presence of spermidine. The dramatic increase in the affinity of poly(dA-dC).poly(dG-dT) for estrogen receptor in the presence of polyamines might be a result of the conversion of the polynucleotide to the left-handed Z-DNA form. These results show that polyamines are capable of participating in estrogenic regulation of gene expression by altering the affinity of the receptor for specific DNA sequences.

Estrogen receptor contents (ER), determined by a sucrose gradient method in 250 primary or metastatic breast cancers, were analyzed with respect to the influence of the patient's menopausal status, chemotherapy, and tissue sampling. The incidence of the presence of estrogen receptors (ER+) increased in the order of para-, pre- and postmenopausal status of the patient, suggesting that a tumor occurring at the paramenopausal phase tends to be a hormone-independent type. Chemotherapy did not alter the incidence of ER(+), nor the quantity of ER in metastatic lesions. Multiple biopsies done simultaneously or sequentially in the same patient are consistent in their qualities and quantities of ER, provided histological confirmation and examination of the amount of cellularity are included in the process of interpretation.

Mithramycin suppresses bone resorption. Its effect on the synthesis and release of beta-glucuronidase (a referent for lysosomal enzymes) in mouse calvarial explants was studied in an in vitro culture system. A newly described medium (designated as KT medium) was introduced in this specific study. Mithramycin initially inhibited the release of beta-glucuronidase into the medium and resulted in an ultimate accumulation of this enzyme in the bone. These results suggest that inhibition of bone resorption by mithramycin may be attributed to interference in release of lysosomal enzymes from bone cells.

Tumor estrogen receptor status in women with advanced breast cancer was correlated with clinical response to cytotoxic chemotherapy in a retrospective study. Following an extramural review of the clinical data of 40 patients, 26 responded to chemotherapy (65%). The response rate in 19 receptor-rich tumors was 89% and in 21 receptor-poor tumors, 43% (P < 0.01). The lowest response rate (14%) was observed in seven postmenopausal patients with receptor-poor tumors. Clinical characteristics of patients and variants in chemotherapy programs failed to explain the favorable response of receptor-rich tumors to cytotoxic chemotherapy.

The effect of RNase on the transformation of progesterone receptor from rabbit uterus was studied by density-gradient centrifugation and DNA-cellulose binding assay. The 7S form of the receptor in crude cytosol was RNase sensitive, and converted to the 4S form after RNase treatment. This reaction was prevented by an RNase inhibitor and reversed by the addition of ribosomal RNA. RNase treatment also caused a two-fold increase in the DNA binding of cytosolic receptor, and reduced the time required for heat-induced transformation. However, sucrose-gradient-purified progesterone receptor (7S) did not undergo transformation by warming unless exogenous RNase was added, thereby suggesting that a cytosolic factor, which might be endogenous RNase, is necessary for the heat-induced transformation of progesterone receptor. Furthermore, degradation of the receptors which occurred after prolonged warming at 25 degrees C in the presence of RNase could be prevented by the addition of DNA-cellulose to the reaction mixture. These results indicate that RNA is associated with the 7S form of progesterone receptor, and that its hydrolysis by RNase might be involved in the transformation of this receptor.

The effectiveness of combination therapy with diethylstilbestrol, cyclophosphamide, and 5-fluorouracil (DES + CTx + FU) was compared with DES alone or CTx + FU in 87 postmenopausal women with advanced breast cancer. Therapy was randomized according to the tumor estrogen-receptor (ER) status. In 30 patients with ER-rich tumors and 35 patients with ER-unknown tumors, combination therapy yielded a higher response rate than DES therapy (87% vs. 64% and 59% vs. 23%, respectively). The pooled data from these two groups of patients suggest that the improved response rate from DES + CTx + FU against DES becomes more apparent in patients with visceral involvement (89% vs. 47%) (P less than 0.025) and that patients treated initially with combination therapy (DES + CTx + FU) appeared to have a longer survival than those treated with sequential therapy (DES leads to CTx + FU) (P = 0.06). The survival data in 22 patients with receptor-poor tumors were significantly inferior to those with receptor-rich tumors (P = 0.001). The ER status and presence of visceral metastases are significant factors in the selection of treatment programs.

SummaryHomogenates of tumorous and adjacent non-tumorous colorectal tissues from 18 patients were tested for the activities of blood group synthetic enzymes, namely α-D-galactosyl transferase for B isoantigen and α-N-acetyl-D-galactosamine transferase of A isoantigen.The galactosyl transferase activity in non-tumorous intestinal tissue was high in patients with blood group B, intermediate in group AB and was absent in group A or O. As compared with adjacent non-tumorous tissue, the Gal transferase activity increased in tumors by 1.6- to 6.9-fold in four of five patients with blood group B or AB, and the α-N-acetyl-D-galactosaminyl transferase activity increased in three- of six-tumor tissues from patients of blood group A or AB.The results suggest that the reported losses of ABH isoantigen in colorectal cancer are not due to deficiencies of Gal or GalNAc transferase activities involved in the synthesis of blood group antigens.

The effectiveness of hypophysectomy and tamoxifen in treating advanced breast cancer was compared in a randomized study of 26 patients who had responded to prior oophorectomy or additive hormonal therapy. When patients failed to respond or relapsed from tamoxifen or hypophysectomy, the therapy was crossed over. In this designed sequence, the rate and duration of response observed with tamoxifen or hypophysectomy used as the first regimen were comparable. The results suggest that tamoxifen is effective in the posthypophysectomy phase and the sequence of hypophysectomy followed by tamoxifen in hormone-dependent breast cancer is preferable to achieve a maximal control of the disease.

We studied the growth inhibitory effects of DL-alpha-difluoromethylornithine, and antiestrogens (tamoxifen, 4-hydroxytamoxifen, trioxifene, keoxifene, and LY117018) as single agents and in combinations on the proliferation of a breast cancer cell line, MCF-7. At 0.1 mM difluoromethylornithine, the proliferation of MCF-7 cells was inhibited to 75 +/- 6% of the controls. Treatment of the cells with 0.1 microM 4-hydroxytamoxifen reduced cell growth to 72 +/- 4%. Combination of 0.1 mM difluoromethylornithine and 0.1 microM 4-hydroxytamoxifen reduced cell growth to 38 +/- 5%, indicating additive growth inhibitory effects. Similar additive effects were observed with all 5 antiestrogens in combination with difluoromethylornithine.

By the conventional steroid-binding assay method for receptor, 3% of 1,095 primary breast cancers (or 10.6% of 263 premenopausal tumors) were classified as negative for estrogen receptor (ER), but positive for progesterone receptor (PR). The true ER status in this rare group of tumors was further investigated by the enzyme-immunoassay (EIA) or immunocytochemical (ICA) staining method using monoclonal antibodies H222 and D547. Immunoreactive ER was present in nine ER-/PR+ tumors studied, whereas it was not detectable in nine age-matched ER-/PR- tumors. Immunoreactive ER was also present in 24 ER+ breast cancers studied, and was particularly higher in tumors that were PR+. Measurement of immunoreactive ER by monoclonal antibody method provides certain advantages over the conventional dextran-coated charcoal (DCC) method, especially in ER-/PR+ tumors.

Estrogen receptor status, tumor histology, and the interval between the development of tumors were assessed in 99 patients with bilateral breast cancer. Tumors were first grouped into those simultaneously detected in both breasts or within 12 months of each other (synchronous bilateral breast cancer, of which there were 64) and second, those detected within more than 12 months of each other (asynchronous bilateral breast cancer, of which there were 35). Nineteen percent of all tumors were lobular carcinomas. Overall, the rate of receptor discordance between the two tumors was not significantly different from that previously reported between biopsies of primary tumor and metastases in patients with unilateral breast cancer. Synchronous receptor-positive tumors occurred significantly more frequently than expected, suggesting that the development of the two tumors was influenced by a common mechanism. In patients with asynchronous bilateral breast cancer there was a significantly longer interval between tumors if both were receptor-positive compared with concordant receptor-negative tumors and tumors with discordant receptor status. There was a significant discordance in the receptor status of asynchronous tumors when the histology also differed, indicating that the tumors in this group were likely to be separate primary tumors.

A patient with an unresectable well-differentiated bile duct tumor who survived for 15 yr after biopsy diagnosis is presented. Histologic examination of the tumor revealed bland features of bile duct adenoma despite extensive spread within the liver. Over its subsequent course, the tumor progressively replaced the liver, achieving huge size, although there was no evidence of metastases until shortly before the patient's death. This clinical course was very unusual for either bile duct adenoma or cholangiocarcinoma, but would be more characteristic of another tumor of intrahepatic bile duct origin, the biliary cystadenoma. However, this latter diagnosis was excluded with both gross and microscopic pathologic criteria. Evidence is presented to support classification of this tumor as an unusual varient of peripheral cholangiocarcinoma which requires correlation of the clinical and pathologic findings for correct diagnosis.

To determine the correlation between the estrogen-receptor status and responses to chemotherapy or hormonal therapy, we retrospectively analyzed the clinical data of 143 patients with advanced breast cancer. Receptor contents were determined by a sucrose-gradient method and designated arbitrarily as "rich" or "poor." The response rate to chemotherapy was significantly higher in receptor-rich tumors (86 per cent) than in receptor-poor tumors (36 per cent) (P

RAPIDLY growing tumor cells depend on a high rate of pyrimidine synthesis for the generation of RNA and DNA. Fluorouracil is a pyrimidine-base analogue that acts as an antimetabolite to block the s...

Fifty-three patients with advanced lymphocytic lymphoma were randomly assigned to treatment with the combination cyclophosphamide, vincristine, and prednisone (CVP) or the same agents used successively in maximal doses (C-V-P). Complete remissions occurred in 68% with CVP and 48% with C-V-P. For patients with nodular lymphoma, the complete remission rate was 81% with CVP and 46% with C-V-P. In patients with diffuse lymphoma a complete remission rate of 50% was obtained with both regimens. The median duration of response was longer for patients who obtained complete remission with CVP (37+ months) than for those entering remission with C-V-P (25+ months). More patients treated with CVP still survive. Current results suggest that CVP is a better induction regime than C-V-P in patients with nodular lymphoma. However, in patients with diffuse lymphocytic lymphoma, neither regimen results in more than 50% complete remissions or significant numbers of prolonged responses. More effective therapy is needed.

We randomized 81 postmenopausal women with advanced breast cancer, whose tumors were rich in estrogen receptors or of unknown estrogen-receptor status, to receive either estrogen therapy alone or estrogen therapy combined with chemotherapy. An additional 31 patients, whose tumors were poor in estrogen receptors, were randomized to receive either chemotherapy alone or estrogen combined with chemotherapy. The median duration of follow-up was 87 months. In the receptor-rich group, the survival of the 21 patients receiving combined therapy was significantly longer than that of 19 patients receiving estrogen as initial therapy (followed by chemotherapy after failure or relapse). The median survivals were 72 and 29 months, respectively (P = 0.05 by the generalized Wilcoxon method). Among 41 patients with tumors of unknown receptor status, a survival advantage from combined therapy over chemotherapy was seen in the first two years and then disappeared. The survival in 31 patients with receptor-poor tumors was uniformly short regardless of the therapeutic method. We conclude that combined therapy offers a survival advantage in postmenopausal patients with receptor-rich tumors.

Immunoassayable insulin was elevated in the plasma and in the primary and metastatic lesions of a patient with carcinoma of the cervix. The release of insulin from the tumor tissue could have been the important factor in precipitating acute episodes of hypoglycemia in this patient.

Hypophysectomy was performed on two male patients with disseminated breast cancer. One of them went through a sequential treatment program and lived 15 years after the initial mastectomy. An objective remission lasting 13 months was observed following hypophysectomy. The other patient is still in remission 7 months after hypophysectomy. Both of them had a previous response to orchiectomy. The response of six other patients reported in the literature was reviewed. From these preliminary results, hypophysectomy could be considered as an effective procedure in the further control of this disease. A sequential therapy for advanced male breast cancer is proposed. Although estrogen therapy may have antitumor activity in male breast cancer, one patient had an apparent stimulation of skin lesions from administered estrogen.

Cytosols from breast cancers were measured for estrogen receptor (ER) and thymidine kinase (TK) activity. There was no correlation between ER and TK in 137 primary breast cancers studied. The results of TK from 57 metastatic breast cancers were correlated with the response or failure to subsequent hormonal therapy or chemotherapy. TK did not predict the responses to hormonal therapy in 12 patients. Of the 45 patients treated with chemotherapies, 13 of 15 tumors with TK over 80 pmol/mg/min responded (86%), while only 4 of the 30 tumors (13%) with TK below 80 pmol/mg/min responded (p less than 0.001). TK appears to be useful in predicting the responses to chemotherapy.

Estrogen receptors (ER) from rat and rabbit uterine cytosol were examined for their sensitivity to ribonuclease (RNase). After RNase treatment, a major part of rabbit uterine ER was converted from the 7S to 3–4S form, and its binding to DNA-cellulose was increased by 40%. Similar treatment on rat uterine ER showed a shift from 7S to 4.5S, and the DNA-cellulose binding was stimulated by 20%. Measurement of endogenous RNase levels showed that lower RNase concentration in rabbit uterine cytosol coincided with larger stimulation of DNA-cellulose binding by exogenous RNase. These results indicate that a major part of 7S ER is susceptible to RNase, and cleavage of bound RNA seems to uncover additional binding sites for DNA. In contrast to the general thinking that 4S to 5S transformation is essential for nuclear binding, we have observed that RNase-treated rat uterine ER did not undergo such a transformation by warming at 25°C, while DNA-cellulose binding of the receptors increased. Thus, temperature activation could occur independent of 4S to 5S transformation.

Summary Studies on the mechanism of activation of estrogen receptor (ER) have led us to identify two classes of receptors. Effect of molybdate on the activation of ER was determined by their affinity for nuclei or DNA-cellulose. Molybdate caused inhibition of nuclear binding with rat uterine ER, but an increase in the binding was observed with rabbit uterine ER. Similar results were observed when DNA-cellulose was used instead of nuclei. To further test the diversity in ER activation, DNA-cellulose binding of ER from 10 primary breast cancer tissues was determined in the presence or absence of molybdate. ER from 8 tissues showed inhibition of binding, one showed an increase, and one was not affected by molybdate. These results indicate the presence of two classes of ER, one whose activation is inhibited by molybdate and another whose activation is either unaffected or stimulated by molybdate. Other differences between rat and rabbit uterine ER are also discussed.

Biological characteristics were assessed in GR mouse mammary tumors during 22 serial transplantations. Although unidirectional progression from hormone dependency to independency was observed, other biological markers such as progesterone receptors, polyploid frequency and thymidine kinase activity demonstrated cyclic phenomena every fourth to sixth transplant generation, suggesting the continued presence of regulatory mechanisms among various cells subpopulations.

The effect of a combination of endocrine and chemical therapies has been studied during the past decade in DMBA-induced rat mammary tumors and advanced human breast cancers. Although interferential effects have been observed between endocrine therapy and chemotherapy in highly hormone-dependent tumors or cell lines, beneficial effects can be achieved from a combination of these two treatment modalities in human breast cancer when the steroid receptor status and the presence or absence of visceral metastases are considered in the selection of treatment programs.

Advanced renal cell carcinoma is relatively resistant to most adequately evaluated chemotherapeutic agents. The combination of CCNU and vinblastine, which has antitumor activity in a frog renal carcinoma model system, has demonstrated activity in initial studies in man. The current study investigated this combination of drugs together with a progestational agent, Delalutin. Seventeen patients with metastatic renal cell carcinoma were treated with CCNU, vinblastine and Delalutin. There were no objective responses. Four patients with stable disease had a mean survival of 18 months compared to 13 patients with progressive disease who had a mean survival of 5 months. The survival of the four patients with stable disease was in large part due to the slowly progressive natural history of their disease.

Mithramycin is effective in the treatment of hypercalcemia. The mechanism of its hypocalcemic effect was studied in six patients with hypercalcemia by serum 85Sr or 45Ca kinetic techniques. Mithramycin was given at two dosage levels (25 or 50μg/kg iv). Mithramycin at the low dosage had little effect on the rate of bone accretion. However, at both dosage levels, mithramycin caused an upward shift of the slope of the specific activity curve, indicating an inhibitory effect on bone resorption. This effect started 6-12 h after a 25-μg/kg dose of mithramycin and lasted from 4-6 days. It appears that mithramycin has a preferential effect on bone resorption. (J Clin Endocrinol Metab 48: 341, 1979)

Stimulation of tumor growth and induced hypercalcemia both may occur during the initiation of estrogen therapy in breast cancer. This study was conducted to determine whether cyclophosphamide (CTX) as an adjuvant to estrogen therapy might (1) prevent induced hypercalcemia or (2) achieve a higher tumoricidal effect during the phase of tumor stimulation. Fifty postmenopausal women with inoperable or recurrent disseminated breast carcinoma were divided into two random groups. Results could be evaluated in 44 patients; 21 received diethylstilbestrol (DES), and 23 received DES plus a 4-week course of cyclophosphamide (DES + CTX). The response rate was 5/21 (24%) in the DES group and 8/23 (35%) in the DES + CTX group (p > 0.05). The median duration of response for both groups was 9 months. The survival rate at 24 months was 52% in the DES group and 25% in the DES + CTX group (p = 0.05). Induced hypercalcemia occurred in 3 patients treated with DES + CTX. Short-term cyclophosphamide adjuvant to estrogen therapy did not prevent induced hypercalcemia nor prolong the duration of response or survival.