Per Briand

Relationship between tumorigenicity, in vitro invasiveness, and plasminogen activator production of human breast cell lines

Two human breast epithelial cell lines (HBL-100, HMT-3522) of non-malignant origin and six (MCF-7, T47-D, ZR-75-1, CAMA-1, BT-20, HMT-3909) of malignant origin have been studied to evaluate whether in vitro invasion and/or secretion of urokinase-type plasminogen activator are related to tumorigenicity in athymic nude mice. Only the six cell lines of malignant origin were tumorigenic. Two of these were invasive in the in vitro assay. These two cell lines were oestrogen receptor negative. Three of the other four cell lines of malignant origin contained oestrogen receptors. The two cell lines of non-malignant origin were neither tumorigenic nor invasive. Thus tumorigenicity was correlated to malignant origin of the cell line whereas invasiveness in vitro was a property of the most undifferentiated cell lines of tumor origin. Secretion of urokinase-type plasminogen activator was neither correlated to tumorigenicity nor to invasion in vitro nor to malignancy of tissue origin.

Effect of antiestrogens and aromatase inhibitor on basal growth of the human breast cancer cell line MCF-7 in serum-free medium.

Antiestrogens are efficient inhibitors of estrogen-mediated growth of human breast cancer. Besides inhibiting estradiol-stimulated growth, antiestrogens may have a direct growth-inhibitory effect on estrogen receptor (ER) positive cells and thus be more efficient than aromatase inhibitors, which will only abrogate estrogen-dependent tumor growth. To address this issue, we have used the human breast cancer cell line MCF-7/S9 as a model system which is maintained in a chemically defined medium without serum and estrogen. The addition of estradiol results in an increase in cell growth rate. Thus, the MCF-7/S9 cell line is estrogen-responsive but not estrogen-dependent. Three different types of antiestrogens, namely tamoxifen, ICI 182,780 and EM-652 were found to exert a significant and dose-dependent inhibition of basal growth of MCF-7/S9 cells. The growth-inhibitory effect of the three antiestrogens was prevented by simultaneous estradiol treatment. Antiestrogen treatment also reduced the basal pS2 mRNA expression level, thus indicating spontaneous estrogenic activity in the cells. However, treatment with the aromatase inhibitor had no effect on basal cell growth, excluding that endogenous estrogen synthesis is involved in basal growth. These data demonstrate that in addition to their estrogen antagonistic effect, antiestrogens have a direct growth-inhibitory effect which is ER-mediated. Consequently, in the subset of ER positive breast cancer patients with estrogen-independent tumor growth, antiestrogen therapy may be superior to treatment with aromatase inhibitors which only inhibit estrogen formation but do not affect cancer cell growth in the absence of estrogens.

Cytogenetic analysis of in vitro karyotype evolution in a cell line established from nonmalignant human mammary epithelium.

Abstract Cytogenetic analysis was carried out on six passages (pass. 16, 19, 21, 25, 34, and 47) of the nontumorigenic epithelial cell line, HMT-3522, established from a fibrocystic lesion of the human breast. Minor chromosome abnormalities were present in the first passage (pass. 16) available for study, and limited cytogenetic progression was observed during the in vitro growth. A modal chromosome number of 45 chromosomes was found in all passages. Each passage contained 4–5 marker chromosomes. Three markers were consistently present in all passages studied. During in vitro growth two markers were gained and two markers were lost from the stemline karyotype. The two latest passages studied had identical karyotypes: 45,XX, del(1)(q44→p32:),t(5;14)(14p13→14q32::5q22→5q35),t(6;8;12;17)(8p23→8q24::6p21.1→6p23;12q24→12p13::6p23→6p25;17p13→17q25::6q11→6q27). The present study demonstrates chromosome abnormalities and karyotypic evolution in a nontumorigenic (in nude mice) and noninvasive (in vitro tested) cell line established from nonmalignant epithelial breast tissue. The results are discussed in relation to gene amplification, double minutes and oncogene localization.

An in vitro model of human breast carcinogenesis: epigenetic aspects

A review is given of 12 years of research on a human breast epithelial cell line, HMT-3522, which has undergone malignant transformation in vitro without being exposed to known carcinogenic agents. Epigenetic aspects of the malignant transformation have been considered and the results have been viewed in a clinical context. It has been concluded that the history and characteristics of the cell line resembles the comedocarcinoma of the human breast. It is hypothesized that progression from benign lesion to comedo in situ carcinoma and invasive carcinoma occurs if low levels of epidermal growth factor are prevailing in the microenvironment. Our data also suggest that breast cancer developed under high epidermal growth factor receptor activity is estrogen receptor negative, while suppression of epidermal growth factor receptor activity promotes estrogen responsive breast cancer.

Induction of cytochrome P-450IA1 gene expression in human breast tumour cell lines

The induction of cytochrome P-450IA1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was studied in eight human breast tumour cell lines. The cells were treated with various concentrations of TCDD for 24 h, and total RNA was isolated. The level of P-450IA1 RNA induced by 1 nM TCDD followed the order: MCF-7 greater than T47-D greater than ZR-75-1 greater than 3909 greater than 3522. AL-1, BT-20 and CAMA-1 did not respond to TCDD at the concentrations used. Northern blot analysis revealed 2 bands at 2.7 and 2.0 Kb, respectively, with the larger band being 6-fold more intense. The ratio was not changed by the TCDD treatment. TCDD induction did not change the benzo[a]pyrene-7,8-diol (BP-7,8-diol) metabolite profile compared with control cells, when cells were incubated with [3H]BP-7,8-diol for 24 h following the treatment with TCDD. These results demonstrate that different breast tumour cell lines vary greatly with respect to the basal expression levels of P-450IA1 RNA and its inducibility by TCDD. Furthermore, TCDD treatment does not change the relative distribution of BP-7,8-diol metabolites.

Molecular cytogenetic analysis of a nontumorigenic human breast epithelial cell line that eventually turns tumorigenic: Validation of an analytical approach combining karyotyping, comparative genomic hybridization, chromosome painting, and single-locus fluorescence in situ hybridization

The immortalized, nontumorigenic human breast epithelial cell line HMT‐3522 has been used as a model for premalignant and, eventually, malignant development. During cultivation, the karyotype evolution was followed. At an early stage, a very long constant phase showed a near‐diploid karyotype, with only five marker chromosomes. DNA from this phase was used for comparative genomic hybridization (CGH) analysis, confirming a previously known MYC amplification, and the integration sites were subsequently determined by single‐locus fluorescence in situ hybridization (FISH). Furthermore, gains of 5q22‐qter and 20q11‐qter and deletion of most of chromosome 6 (6p23‐qter) were detected by CGH. Because of uncertainty about some of the indicated changes, including a deletion of 1p35‐pter, the CGH findings were investigated more closely by chromosome painting, leading to a revision of the karyotype: 45,XX,del(1)(p35),−6,dup(8)(pter→qter::qter→q24),der(12) t(6;12)(p23;p13),der(14)t(5;14)(q22;q32.3),der(17)t(8;17;20)(17pter→17q25::8qter→8q23::8q24→8qter::8q24→8qter::8q23→8q24.1::20q11→20qter). Some karyotypic changes were confirmed by CGH; others had to be revised; and, in the 1p35 region, classical cytogenetics seems superior to CGH. However, CGH revealed a karyotypically unsuspected dup(20q) that might be of special relevance to breast tumor initiation or progression. Our study confirms that CGH is supplementary to current technologies, e.g., karyotyping and Southern analysis, but cannot replace them. In addition, our cell line turned out to be an excellent model for comparison among the different methods. The results imply that future cytogenetic analyses of complex karyotypes should be based on a combination of karyotyping, CGH, and FISH. Genes Chromosom. Cancer 20:30–37, 1997.

Comparison of the immunocytochemical assay (ER-ICA) and the biochemical assay for estrogen receptor in human breast cancer cell lines

Estrogen receptor content of 8 cell lines from non-malignant human breast tissues was determined by an immunocytochemical assay using the Abbott ER-ICA monoclonal kit. The results were in accordance with those obtained by the conventional radiochemical (DCC) assay. Primary cultures of breast tissues are suggested as an important field for in situ application of the ER-ICA.

Complete Loss of Wild-Type TP53 in a Nontransformed Human Epithelial Cell Line Is Preceded by a Phase During Which a Heterozygous TP53 Mutant Effectively Outgrows the Homozygous Wild-Type Cells

HMT-3522 is a spontaneously immortalized cell line derived from a fibrocystic breast lesion. After continuous accumulation of genetic changes, the cell line was transformed from a nontumorigenic to a malignant phenotype. One of the earliest genetic aberrations is a missense mutation of codon 179 (His179Asn) in the tumor suppressor gene TP53 leading to outgrowth of a cell type expressing only the mutant form of TP53. In this report, we extend earlier investigations to reveal the genetic background for the evolution from homozygous wild type to hemizygous mutated cells. The status of the TP53 alleles was followed at different stages by fluorescence in situ hybridization (FISH) and allele-specific PCR (ASPCR) on total DNA, as well as flow-sorted chromosomes--taking advantage of a size difference between the two homologues of chromosome 17 that harbor TP53 on 17p. This further allowed us to determine on which of the two chromosomes the mutated allele was located. The results presented here show that the cells have undergone an evolution from homozygous wild type for TP53 to heterozygous (His179Asn mutation in one allele), and finally to a hemizygous mutated state (deletion of the remaining wild-type allele). The finding of a transient period in which heterozygous cells dominate the population before the eventual outgrowth of hemizygous cells strongly indicates that the His179Asn mutation results in a tp53 protein with a dominant negative effect that does not totally abrogate the function of wild type TP53 in vitro.

Reversible inhibitory activity of sera from breast cancer patients on oestrogen: effect on cell proliferation and protein synthesis of the human breast cancer cell line MCF-7

Abstract Sera from foetal calves, newborn calves, athymic mice, and healthy postmenopausal women exert a growth inhibitory effect on the oestrogen receptor positive human breast cancer cell line MCF-7. This inhibitory effect of serum can be abrogated by oestradiol. Serum samples from 22 breast cancer patients were analysed for the amount of inhibitory activity in order to clarify whether regulation of cell proliferation of human breast cancer may occur via a modulation of the inhibitory activity in the patients serum. Twenty of the 22 serum samples showed inhibitory activity and no difference was found in the degree of inhibition. These results do not support the hypothesis that breast cancer cells grow in vivo solely as a function of a reduced level of a serum-borne inhibitory activity; other mechanisms must be involved in the regulation of growth. We have found that MCF-7 cells, the growth of which is inhibited by serum from breast cancer patients, exhibit a reduced synthesis of three secreted proteins, and an increased amount of one protein, a 46K protein. Oestradiol induced cell proliferation is concomitant with stimulation of the synthesis of these three proteins and inhibition of the 46K protein. Regulation of growth of breast cancer may therefore occur via changes in the synthesis of secreted proteins, which exert a regulatory function on cell proliferation.Sera from foetal calves, newborn calves, athymic mice, and healthy postmenopausal women exert a growth inhibitory effect on the oestrogen receptor positive human breast cancer cell line MCF-7. This inhibitory effect of serum can be abrogated by oestradiol. Serum samples from 22 breast cancer patients were analysed for the amount of inhibitory activity in order to clarify whether regulation of cell proliferation of human breast cancer may occur via a modulation of the inhibitory activity in the patients serum. Twenty of the 22 serum samples showed inhibitory activity and no difference was found in the degree of inhibition. These results do not support the hypothesis that breast cancer cells grow in vivo solely as a function of a reduced level of a serum-borne inhibitory activity; other mechanisms must be involved in the regulation of growth. We have found that MCF-7 cells, the growth of which is inhibited by serum from breast cancer patients, exhibit a reduced synthesis of three secreted proteins, and an increased amount of one protein, a 46K protein. Oestradiol induced cell proliferation is concomitant with stimulation of the synthesis of these three proteins and inhibition of the 46K protein. Regulation of growth of breast cancer may therefore occur via changes in the synthesis of secreted proteins, which exert a regulatory function on cell proliferation.

Cell cycle analysis of estrogen stimulated growth of the human breast cancer cell line, MCF-7

The estrogen receptor positive human breast cancer cell line, MCF-7, can be growth inhibited by high concentrations of newborn calf serum (NCS). MCF-7 cells grown with high concentration of NCS can be growth stimulated by 10(-8) M estradiol, and the growth stimulation seems to involve the abolishment of the effect of inhibitory activity in serum. Flow cytometry has been used to determine cell cycle parameters such as distribution of cells in the different phases of the cell cycle and growth fraction. The cell cycle analysis revealed that addition of 10% NCS to cultures grown with 0.5% fetal calf serum (FCS) increased the doubling time by elongating the G1 transit time. Estradiol stimulation occurred through a shortening of the G1 transit time. An effect on growth fraction was observed neither during growth inhibition with high NCS concentration nor during growth stimulation with estradiol. Cells which are growth stimulated by estradiol have an activated estrogen receptor mechanism as indicated by the presence of filled nuclear estrogen receptors and high level of progesterone receptors. We suppose that a possible mechanism for this estrogen stimulation could be induction of synthesis of growth factors which annual the effect of the inhibitory activity present in NCS.