A new human breast cancer cell line, KPL-3C, secretes parathyroid hormone-related protein and produces tumours associated with microcalcifications in nude mice

Abstract

Summary Parathyroid hormone-related protein (PTHrP) is the main cause of humoral hypercalcaemia of malignancy (HHM). We recently established a new human breast cancer cell line, designated KPL-3C, from the malignant effusion of a breast cancer patient with HHM. Morphological, cytogenetic and immunohistochemical analyses indicated that the cell line is derived from human breast cancer. The KPL-3C cells stably secrete immunoreactive PTHrP measured by a two-site immunoradiometric assay, possess both oestrogen and progesterone receptors and are tumorigenic in female nude mice. The addition of phorbol-12-myristate-13-acetate to the medium significantly increased PTHrP secretion from the cells. In contrast, hydrocortisone, medroxyprogesterone acetate and 22-oxacalcitriol decreased PTHrP secretion in a dose-dependent manner. Unexpectedly, a number of microcalcifications were observed in the transplanted tumours. Radiographical examination indicated that the microcalcifications in the tumours are very similar to those commonly observed in human breast cancer. These findings suggest that this KPL-3C cell line may be useful for studying the regulatory mechanisms of PTHrP secretion and the mechanisms that lead to the deposition of microcalcifications in breast cancer.Keywords: breast cancer; hypercalcaemia; cell line; parathyroid hormone-related protein; microcalcification Parathyroid hormone-related protein (PTHrP) is a recently discovered protein sharing strong homology with parathyroid hormone in the N-terminal amino acid sequence as well as biological activity (Suva et al., 1987;Mangin et al., 1988). This protein was originally isolated from human malignant tumours associated with humoral hypercalcaemia. A number of clinical studies indicate that PTHrP is the main cause of HHM (Burtis et al., 1990;Grill et al., 1991;Ratcliffe et al., 1992). In other words, tumour-derived PTHrP acts as a circulating hormone like parathyroid hormone and induces hypercalcaemia. Recently, a series of studies has indicated that PTHrP is commonly expressed in breast cancer and that a higher expression of PTHrP may induce bone metastasis (Southby et al., 1990;Powell et al., 1991;Bundred et al., 1992;Vargus et al., 1992;Bouizar et al., 1993;Kohno et al., 1994a;Kohno et al., 1994b). It is conceivable that the PTHrP secreted by breast cancer cells, which exist in bone marrow, may act as a paracrine effector on osteoclasts, resulting in osteolytic involvement. These findings suggest that the PTHrP secreted by malignant tumours may act as a hormone or paracrine effector in different pathological situations.Microcalcifications are commonly observed in breast cancer tissues (Snyder and Rosen, 1971 al., 1993).We recently established a new human breast cancer cell line, designated KPL-3C, which is derived from the malignant effusion of a breast cancer patient with HHM. Preliminary characterisation of this cell line and the inhibitory effect of steroid hormones on PTHrP secretion are described in the present paper. Materials and methodsThe clinical co...

Parathyroid hormone-related protein secretion is inhibited by oestradiol and stimulated by antioestrogens in KPL-3C human breast cancer cells

“…It has also been suggested that PTHrP may play a role in the development of osteolytic metastasis. In addition, our previous study suggested that PTHrP might play a role in the deposition of microcalcifications in breast cancer tissues (Kurebayashi et al, 1996). Other researchers have suggested certain roles of PTHrP as autocrine and paracrine growth factors in breast cancer cells .…”

“…Recently, we established a novel human breast cancer cell line, KPL-3C, derived from a patient with humoral hypercalcaemia, and preliminary characterization revealed that this cell line possesses both ER and PgR and stably secretes a detectable amount of immunoreactive PTHrP into the culture medium. A glucocorticoid, progestin and vitamin D analogue significantly inhibited PTHrP secretion from KPL-3C cells in a dose-dependent manner (Kurebayashi et al, 1996). This background prompted us to investigate the effects of oestrogen and antioestrogens on PTHrP secretion in this cell line.…”

“…KPL-3C cells were maintained in RPMI-1640 medium supplemented with 5% fetal calf serum. The population doubling time was approximately 72 h at the exponential growth phase (Kurebayashi et al, 1996).…”

“…The detection limit was 0.5 pM, and the coefficients of intra-and interassay variations were not higher than 7.5%. On the basis of our previous report (Kurebayashi et al, 1996), the PTHrP secretion into the medium was defined as follows:…”

“…Because immmunohistochemical, in situ hybridization or polymerase chain reaction analyses have demonstrated that 60% of primary breast cancers express PTHrP (Southby et al, 1990;Bundred et al, 1991;Powell et al, 1991;Birch et al, 1995) and ER expression is one of the most characteristic features of breast cancer, it is conceivable that there might be an oestrogenic regulation of PTHrP production and secretion in breast cancer. Therefore, the regulation of oestrogen and antioestrogens on PTHrP secretion was investigated in this study using a human breast cancer cell line, KPL-3C, which possesses both ER and progesterone receptor (PgR) and stably secretes immunoreactive PTHrP into culture medium (Kurebayashi et al, 1996). In addition, to investigate the relationship between PTHrP secretion and cell 1819 cycle, the effect of oestrogen and antioestrogens on cell cycle progression was also analysed in this cell line.…”