Elevated levels of pro-cathepsin-d in the plasma of breast-cancer patients

Changes in the expression and processing of cathepsin D (CD) have been shown to be associated with cancer invasion and metastasis. However, the value of CD as a prognostic marker remains controversial. Most studies have used immunological methods to measure the mature form of CD, although it is the precursor (pro-CD) that appears to be abnormally secreted by breast cancer cells. A sandwich-type ELISA has been developed that is specific for pro-CD. The assay employs a monoclonal antibody to mature CD as the capture reagent and a rabbit polyclonal to the pro fragment as the detector. The assay is specific for pro-CD and capable of quantitating this antigen in biological samples. Pro-CD levels were measured in plasma samples from 76 breast cancer patients and compared with 36 samples from normal control individuals. The plasmas of breast cancer patients showed elevated levels of pro-CD; 12% were more than two standard deviations above the mean for the normal samples. Immunoblots of the plasma samples using a CD monoclonal antibody revealed a band at the appropriate size for pro-CD that corresponded in intensity with the ELISA results. Affinity adsorption of breast cancer plasmas with pepstatin agarose followed by immunoblot analysis revealed a single protein band that correlated with pro-CD. Only trace amounts were detected in the normal control plasmas. These results demonstrate that cathepsin D is present in the plasma of breast cancer patients primarily in its precursor form and that it may be a useful prognostic indicator.     

Site-selective cyclic AMP analogues are antagonistic to estrogen stimulation of growth and proto-oncogene expression in human breast-cancer cells

Cyclic AMP (cAMP) analogues that selectively bind to either one of the two binding sites of cAMP-dependent protein kinase demonstrate a potent inhibition of the growth stimulated by estrogen in MCF-7 human breast-cancer cells in culture. The site-selective analogues, which are more potent activators of protein kinase than the analogues studied earlier, exhibit growth inhibition at micromolar concentrations. Among the analogues tested, 8-Cl-cAMP (Site I-selective) and N6-benzyl-cAMP (Site 2-selective) are the 2 most potent inhibitors, causing 40-70% inhibition of the estrogen-stimulated growth at 10-20 microM concentrations with no sign of toxicity. 8-Cl-cAMP (1 microM) in combination with N6-benzyl-cAMP (0.5 microM) almost completely blocks estrogen-stimulated growth, demonstrating synergism between the Site 1- and Site 2-selective analogues. The growth inhibition parallels an increase in the R11 cAMP receptor protein with a decrease in the R1 receptor as well as reduction of c-myc and c-ras oncoproteins, whereas growth inhibition by tamoxifen does not affect the levels of the cAMP receptor proteins or the c-myc and c-ras protein levels. Site-selective cAMP analogues are antagonistic to estrogen stimulation of breast-cancer cell growth through a mechanism different from that of tamoxifen.     

Elevated procaspase levels in human melanoma

In this study procaspase expression levels were investigated by Western blotting in a panel of established melanoma cell lines, transformed melanocytic cell lines and normal primary melanocytes. Upstream caspases such as procaspase-8 that contain a death effector domain were found to be overexpressed in transformed melanocytes and melanoma cell lines compared with melanocytes. Heterogeneous levels of procaspase-8 were seen in melanoma cells, including one cell line that completely lacked procaspase-8 expression. Procaspase-10 is generally overexpressed in transformed melanocytes and melanoma cell lines. Expression of the downstream procaspases-3 and -7 was increased in melanoma cells compared with normal melanocytes. Procaspases containing caspase recruitment domains such as procaspase-2 were expressed at similar levels in nearly all the cell lines investigated. Reduced levels of procaspase-1 compared with normal melanocytes were detected in transformed melanocytes and melanoma cell lines. These data indicate that procaspase levels in general increase during the malignant transformation of melanocytic cells.     

Patterns of cyclic AMP binding in normal human breast

Summary Cyclic AMP binding proteins have been measured in normal breast tissue, in a variety of physiological conditions including resting state (69 cases), pregnancy (15 cases), lactation (4 cases), post-lactational involution (6 cases), and prolonged involution (10 cases). Levels varied greatly within the groups but median values were elevated in pregnancy, lactation, and post-lactational involution as compared with the resting and prolonged involutionary states. The proportion of different types of cyclic AMP binding proteins also differed between groups. Resting breast showed approximately equal amounts of type I and II whereas pregnant and post-lactational involuting breast tissue had an increased proportion of type I binding, and the prolonged involutionary state tended to be associated with low type I. Within the subgroup of nulliparous resting breasts, the proportion of type I cyclic AMP binding protein was significantly elevated during the second half of the menstrual cycle compared with the first (p < 0.05). There were also significant positive associations between the level of epithelial proliferation and both total (p < 0.015) and type I (p < 0.002) cyclic AMP binding. This represents the strongest of associations thus far reported between signalling systems and proliferation in the normal breast.     

Increased intracellular cyclic AMP levels suppress the mitogenic responses of human astrocytoma cells to growth factors

Summary It has been shown that the intracellular cAMP levels were decreased in human malignant astrocytomas. On the other hand, various growth factors and their receptors were found to be overexpressed in these tumors. It is therefore intriguing as to whether there is interplay between the two phenomena in the modulation of the astrocytoma cell growth. In a basal medium consisting of 75% DMEM, 25% Ham's F-12 supplemented with 2% FBS, we show that the mitogenic effects of platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) on human astrocytoma cells were suppressed by dibutyryl-cAMP. Dibutyryl-cAMP alone neither potentiated nor inhibited the tumor cell growth. Further studies show that PDGF-induced receptor autophosphorylation in human astrocytoma cells is suppressed by increased intracellular cAMP levels as measured by immunoprecipitation with anti-PDGF receptor and antiphosphotyrosine antibodies. Our results indicate that there is antagonistic interplay between the receptor tyrosine kinase pathway and cAMP-dependent protein kinase pathway in the control of the malignantly transformed glial cells. A reduced cAMP level seen in many human astrocytoma cells may favor their response to growth factor mitogenesis.     

Elevated pretreatment levels of soluble CD8 and Beta-2-microglobulin as indicators of relapse in breast-cancer patients

We measured soluble CD8 ((S)CD8) and microglobulin (beta-2M) in 128 breast cancer patients and in 200 controls by the ELISA method. Patient groups were: Group A-new patients; Group B-patients on follow-up; Group C-patients with metastases. The mean (S)CD8 and beta-2M were significantly higher in patients than in controls ((S)CD8 p<0.01, beta-2M p<0.0001). Both for (S)CD8 and beta-2M, groups A and C had high levels which differed significantly from Group B ((S)CD8 p<0.04; beta-2M p<0.0001). A significant correlation between (S)CD8 and B-2M was observed (r=0.379: p=0.0001). Twenty patients relapsed. In 14/20 (70%) an initial high (S)CD8 and in 10/20 (50%), high beta-2M was observed. High initial CD8 and beta-2M were able to identify 80% of relapsed patients. High (S)CD8 and beta-2M levels are indicative of tumor bulk and are able to identify patients at.     

Primary tumor levels of interleukin-6 in relation to tumor burden in human breast-cancer

To examine whether tissue levels of interleukin 6 (IL-6) are associated with the clinicopathologic status in human breast cancer, immunoreactive IL-6 concentration was measured in tumor extracts of 75 breast cancer patients. IL-6 was detectable in 69 of 75 tumor extracts by enzyme-linked immunosorbent assay, the concentration ranging from 10 to 10.690 pg/mg protein. When breast cancer specimens were categorized into four groups in terms of clinical stage of disease at diagnosis, IL-6 concentration (mean +/- SE) in tissue extracts was significantly higher in stage IV patients (2859 +/- 840 pg/mg protein) than in stage I-III patients (344 +/- 117, 350 +/- 150 and 564 +/- 230 pg/mg protein, respectively). Correlation analyses between IL-6 concentration and clinicopathologic factors showed that tissue levels of IL-6 were significantly higher in patients with distant metastasis compared with those without. Furthermore, IL-6 concentration was significantly higher in tumors of more than 5.0 cm in size as compared to less than 5.0 cm. However, no significant association was found between IL-6 concentration and age, histological type, histological grade, lymph node involvement, or hormone receptor status. These results suggest that primary tumor levels of IL-6 are closely associated with clinical stage in human breast cancer.     

Exogenous gamma-linolenic acid alters hormone stimulated cyclic AMP levels in U937 cells

Polyunsaturated fatty acids are selectively cytotoxic in culture. Incorporation of these fatty acids leads to profound changes in membrane fatty acid composition which in turn may alter the activity of transmembrane receptor/effector systems. In U937 cells, hormone stimulated production of cyclic AMP can be reduced by 30% following incubation with gammalinolenic acid (18:3n-6). It is suggested that β-adrenoceptor number, subtype and adenylyl cyclase stimulation may be regulated by alterations in membrane fatty acid composition as a result of changes in the levels of polyunsaturated fatty acids and alterations in eicosanoid production.     

Role of cyclic AMP in differentiation of human neuroblastoma cells in culture.

The inhibitors of cyclic AMP phosphodiesterase (papaverine and 4-(-3-butoxy-4-methoxybenzyl)-2-imidazolidinone), serum-free medium, and x irradiation caused cell death and neurite formation in human neuroblastoma cells in culture (IMR-32), whereas theophylline was ineffective. Prostaglandin (PG) E1, N6O'2-dibutyryl adenosine 3',5'-cyclic monophosphate (dbcAMP) induced neurites without causing cell lethality. Inhibitors of phosphodiesterase and PGE1 increased the intracellular level of cAMP by about 2- and 4-fold respectively, whereas serum-free medium and x irradiation did not. The combination of PGE1 and phosphodiesterase inhibitor was more effective in causing morphological differentiation and in increasing the cAMP level than the individual agent. Sodium butyrate induced cell death and neurites, probably in part by increasing the cAMP level. cAMP, guanosine 3',5'-cyclic monophosphate, and adenosine had no detectable effect on the growth or morphology of neuroblastoma cells in culture. Adenosine 5'-monophosphate produced cell death without causing neurite formation. DbcAMP, and to a much lesser degree, sodium butyrate increased the tyrosine hydroxylase activity.     

Monoclonal-antibodies to human breast-cancer associated antigens

Eleven murine monoclonal antibodies (MABs) were produced using human mammary cancer (HMC) cell lines as immunogens: None reacted with normal breast tissue, but each had a distinctive pattern of reactivity with HMC and benign proliferative lesions. Two MABs bound to overlapping epitopes of an M(r) 47,000 cell-surface antigen and were endocytosed: nine bound to secretable intracytoplasmic antigens(s). Four of the MABs immunoprecipitate antigens that upon reduction yielded an M(r) 73,000 moiety. None reacted with components of milk. All antigens were sensitive to trypsin, three to periodate oxidation but none to neuraminidase. One cell-surface-localizing MAB investigated, selectively localized in HMC xenografts.     

Effect of prostaglandins and hormones on cyclic AMP formation in rat hepatomas and liver tissue.

The formation of cyclic AMP was studied in normal liver, subcutaneous hepatomas derived from MH1C1 cells, and premalignant liver and primary hepatomas induced by the carcinogens 2-acetylaminofluorene (AAF) and 4-dimethylamino-azobenzene (DAB). While only very slight effects of prostaglandins (PG) were seen in slices of normal liver, all the hepatomas responded strongly to PGE1 and PGE2. The hepatomas also had increase PGE1-sensitive adenylate-cyclase activity. PGF1alpha and PGF2alpha did not increase the cAMP level significantly either in the liver or in the hepatomas. During AAF carcinogenesis the response to PGE1 increased slightly during the carcinogen feeding, and was greatly elevated only in the fully developed hepatomas. This is in contrast to the increase in adrenalin response seen during carcinogenesis, which starts much earlier, and reaches a peak value within 8--10 weeks. It is concluded that various hepatomas have elevated responsiveness to PGE1 and PGE2 as well as to adrenalin, but the course of change in the tissues' ability to respond to these agents during carcinogenesis is very different.     

Desensitization of human breast-cancer cells by interferon-alpha

Continuous exposure of human breast cancer cells, MCF-7, to interferon-alpha (IFN) induces a state.of non-responsiveness termed as desensitization. mRNA 561 is transiently induced by IFN-alpha in MCF-7 cells, peak cytoplasmic levels are reached by six to twelve hours; the mRNA level declines steadily and is reduced to uninduced levels by forty eight hours. Induction of mRNA 561 was used as an index of responsiveness of cells to IFN-alpha and desensitization was characterized in MCF-7 cells and in MCF-7 cells transfected by the v-H-ras oncogene (MCF-7ras). The kinetics and degree of IFN-mediated induction of mRNA 561 was comparable in both the cell lines. Desensitization was observed in MCF-7 cells and not in MCF-7ras. It was a reversible event, requiring de novo protein synthesis as inclusion of cycloheximide inhibited desensitization. The cellular elements that mediate such a phenomena are elicited by IFNs during the initial phases of IFN action and may be polypeptides. The refractory period, the time after which MCF-7 cells become responsive, was determined to be five days. In conclusion, we demonstrate the use of mRNA 561 induction in evaluating desensitization. Inhibition of protein synthesis or transfection with ras blocks desensitization in MCF-7 cells.     

Ability of lymphocytes infiltrating breast-cancer tissue to convert androstenedione

Lymphocytes were isolated from 43 surgical samples of breast cancer after tumor enzyme digestion and Ficoll/Verographine procedure. In all, 23 specimens from lymphocytic-tissue infiltrates were analyzed (in some cases, material from 2 or 3 patients was combined). The ability of tumor-infiltrating lymphocytes (TIL) to convert androstenedione was demonstrated, as evaluated by hard-water release from the androgenic precursor ³H-1β-androstenedione. In material obtained from menopausal women this ability was higher than in the women of reproductive age. A positive correlation was revealed between the level of androstenedione conversion in TIL and aromatase activity in tumor tissue, while no correlation was shown between androstenedione conversion in TIL and percentage of tumor cells in lymphocytic suspension. The data obtained suggest that factors secreted by a neoplasm are able to induce aromatase gene expression in TIL. Int. J. Cancer 77:485–487, 1998. © 1998 Wiley-Liss, Inc.     

Oxytocin inhibits the proliferation of MDA-MB231 human breast-cancer cells via cyclic adenosine monophosphate and protein kinase A

Oxytocin (OT) inhibits the proliferation of breast-cancer cells in vitro via a specific G-coupled receptor. To elucidate the intracellular mechanism involved in this biological effect, different G-coupled receptor mediators have been investigated in untreated and OT-treated MDA-MB231 breast-carcinoma cells. In these cells, after OT treatment, a significant cAMP increase was observed using a radioimmunoassay procedure, whereas the Ca²⁺ (determined with the fluorescent probe fura-2) and the inositol phosphate (determined after cell labeling with myo(2-³H)-inositol) concentrations were not modified, contrary to what has been observed in myometrial and myo-epithelial cells. The PKA inhibitor PKI (6-22) amide reverted the effect of OT, indicating that the anti-proliferative effect of the peptide is strictly related to the cAMP–PKA pathway. OT treatment did not modify tyrosine phosphorylation either. Our results indicate that in breast epithelial cells devoid of contractile activity, cAMP is the intracellular mediator of OT action, whereas the Ca²⁺-phosphoinositide system is not involved. Int. J. Cancer 72:340–344, 1997. © 1997 Wiley-Liss, Inc.     

Allelic loss at chromosome 8p in human breast-cancer

Loss of heterozygosity (LOH) at loci from the short arm of chromosome 8 has been shown to occur in several types of carcinomas. The consensus deletion region has been recently mapped at 8p21-p22, distal to D8S283 and NEFL loci, and proximal to LPL and D8S265 loci. We report LOH at D8S133, a marker located in this consensus region, in a panel of breast tumor samples. LOH at this locus was observed in about 20% of the tumors.