C-ha-ras enhances the neoplastic transformation of human breast epithelial-cells treated with chemical carcinogens

The present study was carried out with the purpose of analyzing the additive effect of c-Ha-ras oncogene on tumorigenesis in human breast epithelial cells (HBEC) treated with chemical carcinogens. A human breast epithelial cell (HBEC) line, MCF-10F, previously treated with dimethylbenz(a) anthracene (DMBA) and benzo(a)pyrene (BP) was used in these studies. The MCF-10F cells, DMBA and/or BP-transformed cells originated from the clones D3-1 and BP1 which were transfected with the plasmid pH06T1 containing the human T24 mutated c-Ha-ras oncogene and termed MCF-10F-Tras, D3-1-Tras and BP1-Tras, respectively. Whereas the c-Ha-ras transfected cells presented altered morphology, increased anchorage independent growth in agar-methocel, invasiveness and tumorigenicity, the MCF-10F cells, the clones D3 and BP1 were not tumorigenic. Importantly, whereas MCF-10F-Tras was slightly tumorigenic, the D3-1-Tras and BP1-Tras transfected cells were 100% tumorigenic in the SCID mice; and the tumors thus obtained were poorly differentiated carcinomas. DNA fingerprinting confirmed that the tumors derived originated from the cell lineage used. It was concluded that c-Ha ras induces an additive effect on the expression of tumorigenesis in human breast epithelial cell line MCF-10F treated with chemical carcinogens. Our work provide a model for analyzing the role of c-Ha-ras in human breast cancer.     

Genome scanning of breast cancers by two-dimensional DNA typing.

We have recently used two-dimensional DNA typing to detect genetic alterations in breast tumours. This method, which is based on size separation in neutral gels and sequence separation in denaturing gradient gels followed by hybridisation analysis with mini- and microsatellite core probes, allows the simultaneous analysis of hundreds of allelic fragments in a very short time. Here we demonstrate the potency of this method for total genome scanning of the tumour genome by analysing a small series of breast cancers. Comparison of tumour and normal DNA from ten breast cancer patients, using two-dimensional DNA typing with four core probes, revealed a considerable number of genomic alterations. In contrast, with Southern blot analysis only a few alterations were observed using the same probes. Most of the changes observed (74%) were deletions (absence of spots in the tumour) while 20% corresponded to amplifications (spots of higher intensity in the tumour) and 5% were new spots (gains). About 10% of the genomic changes detected appeared to occur in the tumours of more than one patient.     

Preparation of suspensions of human cervical epithelial-cells suitable for biochemical, cytochemical and immunocytochemical investigations

A simple gradient centrifuging method that separates epithelial cells from suspensions of material obtained by conventional cervical sampling procedures, is described. Heterogeneous cell suspensions were disaggregated by syringing and placed on a discontinuous density gradient. Following centrifugation up to 5 bands of, cells could be collected. The three central bands of epithelial cells corresponded to a density range between 1.035g/ml and 1.055g/ml. The cell samples so obtained are suitable for biochemical, cytochemical and immunocytochemical investigations.     

Characterization of a h-3 GnRH method for the measurement of GnRH binding-sites in human breast-cancer and breast-cancer cell-lines

The peptide hormone, GnRH has been hypothesized to play a direct antitumoral role in certain kinds of breast cancer. This direct effect is considered to function via high affinity membrane receptors to GnRH that have been demonstrated in tumors. However, no routine assay for GnRH receptors exist limiting further research in elucidating the mechanisms of GnRH action during treatment and both the clinical usefulness of this measure and the development or refinement of GnRH treatments. Part of the problem has been that previously reported procedures require expensive, difficult (for routine clinical laboratories) iodination of specific GnRH analogs. The use of iodinated substrates present the additional problems of radioactive waste disposal and safety of laboratory personal. Further, the use of different analogs complicates the comparison of data between laboratories. We present an assay that utilizes tritiated natural GnRH in order to assay the native receptor binding and reduce both safety problems and cost of assay/waste disposal. The use of nitrocellulose filters treated overnight with 1% BSA resolved the problems of high blank values. Kinetic data demonstrate that this assay is sensitive, accurate and give results comparable with other methods. We also show that the membranes derived from the tumor and the cell cultures may be frozen at -80 degrees C for up to 90 days without affecting the results of the assay.     

Antisense expression for amphiregulin suppresses tumorigenicity of a transformed human breast epithelial cell line

The epidermal growth factor (EFG) family of receptors and their respective ligands play a major role in breast cancer progression and are the targets of new therapeutic approaches. Following immortalization with SV40 T antigen of normal human breast epithelial cells, a transformed variant cell line (NS2T2A1) was selected for its increased tumorigenicity in nude mice. This cell line was shown to have a higher expression of EGF receptors (EGFR) and amphiregulin (AR) when compared to their normal counterparts or less aggressive transformed cells. Dual staining of EGFR and AR was observed in 50-60% of NS2T2A1 cells, while 30-40% cells expressed AR only. To explore the potential tumorigenic role of AR, a 1.1 kb AR cDNA in an antisense orientation was transfected in NS2T2A1 cells. Three clones, selected by hygromycin B, expressed AR antisense RNA (AR AS1, AR AS2 and AR AS3 cell lines) in which AR protein expression was reduced (ranging from about 50 to < 5%). The anchorage-independent growth of AR AS cell lines was reduced to levels ranging from 32.4-6.8% relative to the control cell line transfected with the vector alone. The clones expressing AR antisense RNA showed a reversion of the malignant phenotype when injected in nude mice, since a significant reduction of tumor intake was observed coincident with a significant tumor mass reduction (> 96%). Moreover, intra-tumoral vascularization decreased significantly in tumors derived from AR AS cells (26.7, 70.7 and 50.4% of control). These in vitro and in vivo data reveal the oncogenic nature of AR in transformed breast epithelial cells and imply a role for AR in tumor angiogenesis.     

Removal of arylamine-DNA adducts in cultured epithelial-cells of rat and human urinary-bladder and mammary-gland

Primary mammary and urinary bladder epithelial cells of the rat, and the human urothelial cell line HCV-29 and mammary cell line MCF-10A were incubated for 3 h with 10 mM hydroxyurea and N-hydroxy-N-acetyl-2-aminofluorene, N-hydroxy-N-acetyl-4-aminobiphenyl or N-hydroxy-3,2'-dimethyl-4-aminobiphenyl. DNA adducts of these carcinogens were detected in the nuclei of all cells, except MCF-10A, semi-quantitatively with a novel immunohistochemical-microdensitometric method. The loss of these adducts in these cells was biphasic and was relatively greater between 0 and 24 h than between 24 and 48 h. The half-life of the adducts of all 3 carcinogens was approximately 20 h. The adduct loss is consistent with repair detected by an unscheduled DNA synthesis system reported previously. These results demonstrate that nonacetylated aromatic amine-DNA adducts can be repaired in the target cells for aromatic amine-induced carcinogenesis.     

Cathepsins D,B, and L in transformed human breast epithelial cells

To investigate the regulation of lysosomal enzymes during carcinogenesis, we measured cathepsins (Cats) D, B, and L in MCF-10F, which is a human breast epithelial cell line, and cells evolved after treatment with carcinogen and transfected with c-Ha-ras oncogene. The clones used in this study, MCF-10FTras, D3, D3-1, and D3-1Tras, expressed no estrogen receptors and gradually increased invasive potential, while oncogenetransfected lines were also tumorigenic in SCID mice [16,19]. Cats D, B, and L were determined in the cells and in cell media using enzyme-linked immunosorbent assay (ELISA), specific enzyme activity measurements, and immunocytochemistry. The major intra- and extracellular lysosomal proteinase in these cells was Cat D (30–180 pm/mg), followed by Cat B (2–10 pm/mg) and Cat L (1–5 pm/mg). An inverse relationship between intracellular Cat D levels and invasive potential of carcinogen-treated and c-Ha-ras oncogene-transfected cell lines was observed. No significant changes in extracellular concentration of Cat D precursor in this series of cell lines was observed. Intracellular levels of Cats B and L were unchanged or slightly lower in carcinogentreated D3 and D3-1 cells, as well as in MCF-10FTras. On the other hand, in D3-1Tras cell line, evolving from c-Ha-ras transfected D3-1 line, 3.5 fold and 4.4 fold increases in Cat B and Cat L, respectively, but a 2 fold decrease in Cat D, were observed compared to the parental cell line. Immunocytochemical staining showed a granular, polarized perinuclear and cytoplasmic staining of cathepsins in all cell lines. Cysteine proteinases stained more frequently and more intensely in D3-1Tras compared to other lines, confirming the immunochemical assays. We hypothesize that several molecular events, caused by a carcinogen and an oncogene such as c-Ha-ras, are needed to increase Cat B and Cat L, but not Cat D, expression. Therefore, the cysteine and aspartic lysosomal proteinases are differentially expressed in the breast cell lines with more invasive phenotype.     

Fatty-acid composition of breast and iliac adipose tissue in breast-cancer patients.

In order to determine to what extent the fatty-acid composition of breast adipose tissue is representative of the body-fat composition in breast carcinoma, we compared the fatty-acid composition of breast adipose tissue to that of iliac fat in breast-cancer patients. Triglycerides from the 2 sites were purified by thin-layer chromatography and fatty-acid composition was determined by capillary gas chromatography. Compared with iliac fat, mammary fat was higher in saturated (33.2 +/- 3.9 vs. 24.4 +/- 1.6%; p = 0.0001) and lower in mono-unsaturated (48.0 +/- 2.2 vs. 54.8 +/- 2.7%; p = 0.0001) and poly-unsaturated fatty acids (16.6 +/- 3.7 vs. 18.0 +/- 3.1%; p = 0.0001). A positive correlation was found between the 2 sites for linoleate (r = 0.95; p = 0.0003), alpha-linolenate (r = 0.83; p = 0.01), palmitate (r = 0.78; p = 0.02) and palmitoleate (r = 0.76; p = 0.02). No relationship was observed for stearic and oleic acids. We conclude that breast and iliac fat differ with regard to fatty-acid composition. The interpretation of fatty-acid composition of the body stores in breast-cancer patients, as an indicator of long-term intake of dietary fat, should take into account the sampling site of stored lipids.     

Comparative genomic hybridization of human breast epithelial cells transformed by estrogen and its metabolites

The elevated incidence of breast cancer in women has been associated with prolonged exposure to high levels of estrogens. Our laboratory has demonstrated that treatment of the immortalized human breast epithelial cells MCF-10F with 17beta-estradiol (E2) or its metabolites 4-hydroxy-estradiol (4-OH-E2) and 2-hydroxy-estradiol (2-OH-E2) induces phenotypical changes indicative of neoplastic transformation. The MCF-10F cells treated with E2, 2-OH-E2 and 4-OH-E2 form colonies in agar methocel and lost their ductulogenic capacity in collagen matrix, expressing phenotypes similar to those induced by the carcinogen benz(a)pyrene (BP). To investigate whether these phenotypic changes were associated with genomic changes, MCF-10F cells treated with either E2, 2-OH-E2, or 4-OH-E2 at different doses (0.007 nM, 70 nM and 3.6 microM) were analyzed using a combination of standard G-banding and comparative genomic hybridization (CGH). Whereas no aneuploidy was observed in any of the transformed cells, the CGH revealed instead that only cells treated with 4-OH-E2 at the highest concentration (3.6 microM) exhibited DNA gains at 8q24, 9q34 and 20q13 and losses at 13q21.     

Combined effects of lonidamine and tamoxifen in human breast-cancer cell-lines

The antiproliferative activity of lonidamine, alone or in combination with the antiestrogen tamoxifen, was studied on estrogen receptor-positive and estrogen receptor-negative human breast cancer cell lines. Lonidamine by itself induced an appreciable cytotoxic effect on all five cell lines, with 50% inhibitory concentrations (IC50) ranging from 19.5 to 54 mu M. The combination of lonidamine and tamoxifen, simultaneously administered or in sequence, provided additive effects on the estrogen receptor-negative MCF/DX cell line and a sub-additive interaction in the estrogen receptor-positive MCF7 cells. The negative interference between the two drugs could be ascribed to the marked reduction induced by lonidamine in the expression of estrogen receptor in the MCF7 cells.     

Benign mammary epithelial cells enhance the transformed phenotype of human breast cancer cells

Background  

Recent research has yielded a wealth of data underscoring the key role of the cancer microenvironment, especially immune and stromal cells, in the progression of cancer and the development of metastases. However, the role of adjacent benign epithelial cells, which provide initial cell-cell contacts with cancer cells, in tumor progression has not been thoroughly examined. In this report we addressed the question whether benign MECs alter the transformed phenotype of human breast cancer cells.     

CHANGES IN ACTIVITY OF INSULIN RECEPTOR TYROSINE KINASE AND CHARACTERISTICS OF ITS ENDOGENOUS SUBSTRATE IN TRANSFORMED HUMAN LYMPHOCYTES

The cascade reaction of tyrosine phosphorylation provides an attractive mechanism to regulate the metabolism and growth of normal and transformed cells. Thus, considerable effort has been made to identify the cellular substrates of the tyrosine kinases. In this work, the activity of the Insulin receptor tyrosine kinase and endogenous substrate in transformed lymphocytes were studied. Purified human T lymphocytes incubated with phytohemagglutinin (PHA) for 72 hours served as transformed cells, when labeled with [32P] - orthophosphate it appeared that the insulin-dependent protein kinase activity in the transformed cells increased 9- fold. In search for the physiologically significant substrates by using polyclonal antiphosphotyrosine antibody to immunoprecipitate phosphotyrosine-containing proteins that produced in the intact cell during insulin stimulation, a protein with molecular weight of 45kDa as identified and designated as PP45, which occurred during the 5 minutes response of lymphocytes to insuli     

Mapping of chromosome-3 alterations in human breast-cancer using microsatellite PCR markers - correlation with clinical-variables

Human breast tumours were analyzed with polymorphic microsatellite markers specific to chromosome 3p. Allelic imbalance (AI) was observed in 34% of the tumours. Microsatellite markers from two regions showed higher percentage of imbalance suggesting the presence of tumour suppressor genes or genes important for malignancy. Microsatellite instability was also found, implying errors in DNA replication. No significant correlation was found between AI and conventional prognostic variables. However, a striking correlation was found between AI and tumour S-phase fraction; AI was also significantly correlated with low steroid receptor content. A multivariate model including prognostic variables, showed that AI was without exception a significant prognostic variable; patients having tumours with AI had approximately a four-fold increase in relative risk of death. We conclude that screening for 3p deletions gives prognostic information and further investigations should reveal whether this finding could assist in treatment of the disease.     

Oncoprotein expression and morphological phenotypes of human breast epithelial cells transformed by the c-Ha-ras oncogene

Activated oncogenes have been detected in a variety of malignant tumors and altered expressions of certain genes are known to play a functional role in the cancer process. The chemical carcinogen, BP, and the insertion of c-Ha-ras, induced characteristics of transformed phenotypes in a suitable human breast epithelial cell line. Carcinogen-treated and Ha-ras-transfected cells showed a progression of changes in the morphology, anchorage independent growth, invasiveness and tumorigenicity in SCID mice. Tumor growth occurs after a series of molecular events that parallel morphological changes. The aim of this work was to determine the neoplastic phenotypes following treatment with benzo(a)pyrene (BP) and transfection with c-Ha-ras oncogene changes and PCNA, Neu, ErbB-3 and Cytokeratin 18 protein expression in MCF-10F cells, a spontaneously immortalized human breast epithelial cell line. Protein expression was determined by immunofluorescent staining coupled with confocal microscopy. An increased oncoprotein expression in comparison to MCF-10F cells was observed in PCNA, Neu, ErbB-3 and Cytokeratin 18 protein expression in breast epithelial cells transformed with a chemical carcinogen and/or oncogene transfected that are not present in the MCF-10F. This in vitro cancer model can be used as a valuable model in the study of breast carcinogenesis.     

Requirement for isoprenoid-dependent posttranslational modifications in the cell-cycle progression of human breast-cancer cells

Treatment with HMG CoA reductase inhibitors, i.e. 25-hydroxycholesterol and mevinolin, inhibited cell growth of the human breast cancer cell line MDA231 in a cell cycle-specific manner by blocking progression through G1. Since 25-hydroxycholesterol, as distinguished from mevinolin, also inhibits steps in mevalonate metabolism, exogenous mevalonate failed to overcome the 25-hydroxycholesterol-induced block. Using 25-hydroxycholesterol we investigated whether protein isoprenylation or protein glycosylation is rate-limiting for G1-progression in MDA231. We thereby found that 25-hydroxycholesterol was efficient in inhibiting N-linked glycosylation, measured by determining the glucosamine incorporation into cellular proteins. In contrast, 25-hydroxycholesterol did not depress the level of protein isoprenylation, measured as incorporation of mevalonate into cellular proteins. Furthermore, tunicamycin (an inhibitor of N-linked glycosylation) inhibited G1-progression of MDA231 in a similar way to 25-hydroxycholesterol. Addition of trans-trans farnesol, which inhibits protein isoprenylation, did not result in any inhibitory effects on MDA231 growth. Our data suggest that N-linked protein glycosylation is rate-limiting in the isoprenoid-regulated cell cycle of human breast cancer cells.     

A rapid in vitro method for measuring cell proliferation in human breast cancer.

A method has been developed for studying in vitro the cell proliferation kinetics of human breast cancer, Surgical specimens from primary tumors were studied in 56 patients. Viable cell suspensions for assay were obtained by the dissociation of tumor tissue with collagenase. Mean Labeling indices of 2.43 +/- S.D 2.05 and 4.48 +/- S.D. 3.73, respectiviely, were found after incubation with 3HTdR for 2 hours and 24 hours. Mean S-times of 21.9 +/- S.D. 4.3 hours were estimated by 3H and 14C-TdR double-labeling. The kinetic data have been validated by parallel labeling studies in vivo and in vitro in four patients. The processing of autoradiographs using gold latensification provided slides for kinetic analysis within 3 days. The assay offers a method that is useful in the planning and monitoring of drug therapy.     

Expression of endogenous granzyme B in a subset of human primary breast carcinomas

Granzyme B (GrB) is the prototypic member of a serine protease family primarily used by cytotoxic lymphocytes to kill target cells. We report here that, by immunohistochemical staining of paraffin-embedded tumour sections, GrB protein was unexpectedly detected in malignant cells of a subset of breast cancers and their adjacent reactive endothelial and mesenchymal cells in which endogenous retinoblastoma protein (pRB) is overexpressed. The identity of the endogenous GrB was further confirmed experimentally in RB-deficient breast carcinoma cell culture upon overexpression of ectopic pRB. Our finding extends the recent paradigm-shifting trend for a more diverse biological role of granzyme B, and might provide a rational basis for exploring its potential prognostic value in a variety of human cancers.     

Comparison between concentrations of trace elements in normal and neoplastic human breast tissue

Histologically normal and neoplastic human breast tissues obtained from 25 patients at the time of mastectomy were homogenized (200 mg/ml) in distilled water and 5-microliter aliquots dried on Formvar films for trace element analysis by energy-dispersive X-ray fluorescence. The elements measured were calcium, vanadium, copper, zinc, iron, chromium, manganese, nickel, selenium, molybdenum, bromine, rubidium, strontium, mercury, arsenic, and lead. In general, significantly large increases (p less than 0.001) in calcium, vanadium, copper, zinc, selenium, and rubidium were found in breast tumors, with a less significant increase (p less than 0.05) for nickel. When a comparison was made between histologically normal and neoplastic tissues from the same individual, zinc and rubidium were found to be consistently higher in the tumor, whereas calcium, copper, and vanadium levels varied from normal to high. In no instance were the tissue changes in calcium, copper, zinc, or rubidium reflected in the blood levels, which were within normal limits. The distribution of calcium, copper, and zinc in urine varied among individuals with primary tumors; however, rubidium levels tended to be consistently elevated. An attempt is being made to correlate these various differences with the extent of the primary disease at the time of surgery, the postoperative tumor-free interval, and subsequent therapy.