Urs Regenass

Cytogenetics of breast cancer

Chromosome counts were performed on 1,100 cells from 17 malignant breast carcinomas and on 168 cells of four normal tissue samples after amethopterin treatment and G-banding. Karyotypes were established from 216 cells of 11 tumor-derived cultures and from 47 cells of four nonmalignant tissue-derived cultures. Karyotypes of cells from nonmalignant samples showed a normal diploid chromosomal constitution with no consistent loss or gain of a specific chromosome. Structural chromosomal abnormalities were not observed. Tumor-derived cultures could be distinguished from normal cultures on the basis of a significantly increased incidence of numerical changes and structural chromosomal aberrations. In nine of 11 tumor-derived cultures, numerically normal cells were shown to be pseudodiploid, with frequencies ranging to 43% (mean, 13.2%) of the diploid cells. In agreement with previous reports, cytogenetic analyses showed predominantly diploid cells. Clonal numerical changes of chromosomes 17, 18, 20, and 21 could be detected in three tumor samples. Clonal structural abnormalities could be observed in two of 11 analyzed tumours. A t(6;12)(p21;p13) and an enlarged chromosome 7 (7q+) were found in a patient with invasive ductal carcinoma. An inversion of chromosome 7 [inv(7)(q11.2q32)] was observed in one case, also diagnosed as invasive ductal carcinoma. The significance of these findings in relation to clinical data is discussed.

Reversal of multidrug resistance by the staurosporine derivatives CGP 41251 and CGP 42700

It has been shown previously that the staurosporine derivative CGP 41251, a specific inhibitor of protein kinase C (IC50 = 50 nM), exhibits antitumor activity and reverses mdr1‐mediated multidrug resistance. At present, the compound is evaluated as an anticancer drug in clinical phase I trials. We compared the effects of CGP 41251 with CGP 42700, another staurosporine derivative, which exhibits low protein kinase C inhibiting activity (IC50 = >100 μM). We found that in contrast to CGP 41251, CGP 42700 does not show antiproliferative activity in HeLa and KB cells in tissue culture (up to a concentration of 10 μM). We compared both compounds for their ability to reverse mdr1‐mediated resistance in KB‐C1 and in HeLa‐MDR1 cells (transfected with the mdr1 gene). CGP 42700 is able to reverse mdr1‐mediated resistance to a similar extent as CGP 41251. The intracellular accumulation of rhodamine 123 in KB‐C1 cells following pretreatment with CGP 41251 for 30 min was higher than that following treatment with CGP 42700 if determined in medium without serum. However, quantitation of rhodamine efflux in an ex vivo assay using human CD8+ cells in serum showed that CGP 42700 is more effective in inhibiting the efflux of rhodamine 123 than CGP 41251. We conclude from our results that (1) CGP 42700 is more effective in reversal of multidrug resistance in serum than CGP 41251, indicating that the compound may be useful for treatment of patients, and (2) CGP 42700 does not inhibit protein kinase C and cell proliferation and, therefore, may be less toxic and elicit less side effects in humans than other chemosensitizers. Int. J. Cancer 77:64–69, 1998.© 1998 Wiley‐Liss, Inc.

Use of 4-fluoro-l-ornithine to monitor metabolic flux through the polyamine biosynthetic pathway

The mechanistic effectiveness of various polyamine analogs and enzyme inhibitors is typically determined by their ability to deplete intracellular polyamine pools. In this study, we describe an assay that may prove useful in augmenting this relatively static assessment of drug action. The assay relies upon the substitution of 4-fluoro-L-ornithine (Fl-Orn) for ornithine as a polyamine precursor to provide a means to measure metabolic flux through polyamine pools. At concentrations up to 500 microM, the analog did not inhibit the growth of L1210 murine leukemia cells during incubations of up to 72 hr. Using HPLC, the analog was processed metabolically over time to what was deduced to be 2-fluoroputrescine, 6-fluorospermidine and 6-fluorospermine. The relative proportion of fluorinated polyamine analog to the natural polyamine increased with time and Fl-Orn concentration. The sum of the two was found to be nearly identical to the respective polyamine pool of control cells exposed instead to 500 microM ornithine. This indicates that Fl-Orn was recognized and utilized as a precursor at a rate very similar to that of ornithine itself. Using L1210 cells at different stages of cell growth, it was determined that the metabolic flux through the pools, as indicated by the rate of appearance of individual fluorinated polyamine species, reflected the proliferation status of the cells--non-growing cells failed to incorporate the analog. Likewise, in cell types with varying polyamine pool profiles, such as polyamine enzyme overproducers or those with constitutively different spermidine of spermine ratios, the incorporation of the fluorinated analogs into pools was found to be proportional to the size to the natural polyamine pool. In cells treated with inhibitors of S-adenosylmethionine decarboxylase, Fl-Orn incorporation indicated a total blockade of polyamine synthesis at that enzyme site. Overall, the Fl-Orn assay has demonstrated that polyamine pool profiles generally reflect the rate of flux through the pathway in proliferating cells, suggesting that most intracellular polyamines are freely exchangeable with those undergoing metabolic flux.

Pharmacological properties of the ornithine decarboxylase inhibitor 3-aminooxy-1-propanamine and several structural analogues

Analogues of 3-aminooxy-1-propanamine proved to be highly potent and selective inhibitors of ornithine decarboxylase (ODC). The compounds competed with ornithine for the substrate binding site of ODC, but resulted in progressive and apparently irreversible inactivation of the enzyme. Diamine oxidase was inhibited by these compounds to a lesser extent than ODC; the compounds were not metabolized by this enzyme. Several derivatives were growth-inhibitory for human T24 cells and for other mammalian cells, the most active compound being 3-aminooxy-2-fluoro-1-propanamine (AFPA). Growth-arrested cells were largely depleted of putrescine and spermidine. Cellular growth arrest could be antagonized by supplementation with spermidine. Selection for resistance against AFPA led to cells with amplified ODC genes and overexpression of the message. Some of the derivatives were tumoristatic at well-tolerated doses in mice bearing solid T24 tumours. The antiproliferative activity of these compounds appears to be mediated by polyamine depletion.

Immunolocalization of cytoskeletal elements in human mammary epithelial cells.

The expression of cytokeratins, vimentin and actin was analysed by immunohistochemical methods in primary cultures derived from breast carcinomas and non-malignant breast tissues, in tissue sections and in permanent breast cancer cell lines. A polyclonal antibody specific for cytokeratins bound to the majority of cells in all primary cultures. Cells positive for simple-epithelial cytokeratins were less frequent in primary cultures derived from malignant tissues than in cultures derived from non-malignant tissues. Established cell lines MCF-7 and BT-20 expressed high amounts of simple-epithelial cytokeratins. All outgrowing cells in primary cultures expressed high amounts of vimentin whereas vimentin expression in established carcinoma lines was variable but generally low. When paraffin sections of the original mammary tissues were analysed, epithelial cells were devoid of vimentin as expected. Anti-actin antibody decorated fine fibres in all cells of primary cultures in contrast to the diffuse staining found in established mammary carcinoma cell lines.

Involvement of protein kinase C in the growth regulation of human breast cancer cells

The relationship between growth of breast cancer and endocrine regulatory mechanisms is well established [1, 2, 3, 4, 5, 6, 7]. Numerous studies provided evidence for a distinct and important role of estrogen and progesterone and their receptors (ER and PR) in the growth regulation of human breast cancer (HBC) [1, 2, 3, 4, 5, 6, 7]. At present there is no unifying hypothesis about specific alterations that lead to the initiation and progression of HBC [5, 6, 7]. However, it is clear that human breast cancer progresses from an estrogenresponsive (hormone-dependent) ER+ tumor to a more aggressive estrogenunresponsive (hormone-independent) ER- phenotype that is less or poorly amenable to endocrine therapy [1, 2, 3, 4, 5, 6, 7]. Since only few hormone-independent HBC tumors lack the ER, the loss of the ER cannot explain the lack of antiestrogen sensitivity [1, 2, 3, 4, 5, 6, 7]. Despite many studies, the molecular mechanisms by which steroid hormones regulate the growth of both normal mammary and tumor cells remain elusive. It has been proposed that the escape from estrogen-dependent growth of HBC cells is due, at least in part, to alterations in their autocrine and/or paracrine growth mechanisms [16, 17, 18, 19, 20, 21, 22, 23]. Consequently, disordered secretion of growth factors (GFs) from HBC cells and/or surrounding stromal and other cells may result in a permanent activation of intracellular signaling pathways contributing in the bypass of the estrogen-dependent growth control of HBC cells and hence progression of the disease.

In Vitro Cultures of Epithelial Cells from Healthy Breast Tissues and Cells from Breast Carcinomas

Much of the knowledge concerning the mechanisms involved in the growth regulation of breast cancer comes from the use of cultured cells, particularly cell lines (Lippman et al. 1986; Salomon et al. 1986). During the last few years, techniques have been described which allow the growth directly from patients’ tissue of normal human breast epithelial cells (Stampfer et al. 1980; Stampfer 1982; Biran et al. 1983; Taylor-Papadimitriou et al. 1980; Hammond et al. 1984; Briand et al. 1987; Soule and McGrath 1986; Emerman et al. 1987) and of human mammary carcinoma-derived cells (Smith et al. 1981; Simon et al. 1984; Emerman et al. 1987; Petersen and Van Deurs 1987).

A derivative of staurosporine (CGP 41:251) shows selectivity for PKC inhibition and in vitro antiproliferative effects as well as in vivo antitumor activity. Int. J. Cancer,43, 851–866 (1989)

MEYER, T., REGENASS, U., FABBRO, D., ALTERI, E., RÖSEL, J., MÜLLER, M., CARAVATTI , G. and MATTER, A., A derivative of staurosporine (CGP 41:251) shows selectivity for PKC inhibition and i vitro antiproliferative effects as well as in vivoantitumor activity.Int. J. Cancer,43,851–866 (1989) are not considered reliable. The authors wish to retract these data. In addition, thein vivoexperiments described in the article