PCR detection of cytokines in normal human and pagetic osteoblast-like cells.

We investigated the expression of cytokine transcripts in osteoblast-like cells derived from explants of pagetic and normal bone. A reverse transcription-linked PCR was used that allowed the simultaneous analysis of a range of cytokines. Normal osteoblast-like cells were found to contain the transcripts for IL-1 beta, IL-6, and TGF-beta 1. For the first time we detected in bone cells the two other mammalian isoforms of TGF-beta, beta 2, and beta 3. Furthermore, we have also identified mRNA for IL-3 and the novel chemotactic factor, IL-8. Using this sensitive technique it was not possible to detect mRNA for IL-1 alpha, IL-2, IL-4, IL-5, IL-7, TNF-alpha, or interferon-gamma. The human osteosarcoma cell line Saos-2 also showed a similar pattern of expression of these cytokines to primary osteoblast-like cells, with the exception that TNF-alpha was also identified. Cells isolated from pagetic bone showed essentially the same profile of cytokine expression as normal bone except that TNF-alpha was also detected in two of four samples. The cytokine profile of successive populations of cells harvested from one explant culture at 9, 22, and 57 days showed a consistent pattern of cytokine expression, demonstrating the phenotypic stability of the osteoblast-like cells in long-term cultures.

Transforming growth factor-beta 1, beta 2, and beta 3, urokinase and parathyroid hormone-related peptide expression in 8701-BC breast cancer cells and clones.

8701-BC is a recently characterized cell line isolated from a primary ductal infiltrating carcinoma of the breast (d.i.c.), showing some pleomorphism in cell microanatomy at an ultrastructural level. We have obtained different sublines of 8701-BC cells by cloning in soft agar at different concentrations (0.3% and 0.6%), and we have characterized the cloned lines by some morphological and growth parameters. 8701-BC cells and clones have been submitted to analysis by reverse transcriptase-linked polymerase chain reaction to detect mRNAs of various cytokines (transforming growth factor-beta s, tumour necrosis factors, interleukin 1s, interleukin 6, parathyroid hormone-related peptide, gamma interferon) and of urokinase, which are bioactive molecules commonly involved in cell-cell and cell-stroma interactions at primary and/or secondary sites of invasion. The aims of the present investigation were to determine: (a) if the corresponding genes are active in 8701-BC cell line and (b) if the sublines tested exhibit transcriptional heterogeneity. The results obtained show that 8701-BC cells express transcripts of transforming growth factor-beta s, urokinase and parathyroid hormone-related peptide (PTHrP), the latter product being responsible for the cancer-associated humoral hypercalcemic syndrome. Moreover, while the first two mRNAs are detectable in all the sublines tested, PTHrP is expressed almost uniquely by the clones isolated in 0.6% agar which exhibit a peculiar morphological appearance, a higher growth rate and a more active invasive behaviour in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Extracellular nucleotides stimulate proliferation in MCF-7 breast cancer cells via P2-purinoceptors.

Nucleotides such as ATP can act as extracellular effector molecules by interaction with specific cellular receptors known as P2-purinoceptors. Recently, we cloned the human P2U purinoceptor from osteoclastoma and demonstrated its expression in skeletal tissues. In the current study we have investigated the expression of P2U purinoceptors in human breast tumour cell lines and examined functional effects of extracellular nucleotides on these cells. By reverse transcription-linked polymerase chain reaction (RT-PCR) the expression of mRNA for P2U purinoceptors was demonstrated in four human breast cancer cell lines, Hs578T, MCF-7, SK-Br3 and T47-D. In MCF-7 cells, extracellular ATP (1-100 microM) elevated intracellular free calcium concentration [Ca2+]i, indicating that these cells express functional P2-purinoceptors. UTP elevated [Ca2+]i in an identical manner to ATP, whereas 2-methylthioATP was completely ineffective, and ADP only partially effective. This pharmacological profile suggests that the P2U subtype may be the only P2-purinoceptor expressed by these cells. The functional significance of P2U purinoceptor expression by MCF-7 cells was investigated by analysing the effects of extracellular ATP on cell proliferation. The slowly hydrolysed analogue of ATP, ATPgammaS (which was also shown to elevate [Ca2+]i), induced proliferation of MCF-7 cells when added daily to serum-free cultures over a period of 3 days. ATPgammaS-induced proliferation was demonstrated by three separate methods, detection by scintillation counting of [3H]thymidine incorporation, immunocytochemical detection of 5-bromo-2-deoxyuridine incorporation and direct counting of cell numbers. These data suggest that ATP, possibly released at sites of tissue injury or inflammation, may be capable of growth factor action in promotion of tumour proliferation or progression.

Cytokine expression by cultured osteoblasts from patients with osteoporotic fractures.

Human osteoblasts were derived in culture from explants of bone from patients who had recently suffered osteoporotic fractures and from patients with no evidence of osteoporosis. The expression of cytokine mRNA in these osteoblasts was subsequently determined by reverse transcriptase-linked polymerase chain reaction (RT-PCR). We have detected mRNA for IL-1beta, IL-3, IL-6, IL-8, TNF-alpha and -beta, and the three TGF-beta isoforms in the cells. The profile of cytokines expressed by osteoblasts derived from patients with osteoporotic fractures was consistent with profiles observed in osteoblasts derived from patients with no evidence of reduced bone mass--the latter included children undergoing corrective surgery and adult subjects ranging from 31 to 80 years undergoing elective surgery for osteoarthritis and other bone pathologies.

Hypercalcaemia due to parathyroid hormone-related protein: long-term circulating levels may not reflect tumour activity.

Parathyroid hormone-related protein is responsible for the hypercalcaemia caused by many tumours. Measurement of parathyroid hormone-related protein is becoming more accessible with the introduction of commercial assays. We report a case of hypercalcaemia of malignancy secondary to parathyroid hormone-related protein in a woman with renal carcinoma. The parathyroid hormone-related protein was assayed using a new immunoradiometric assay. We demonstrated an initial fall in parathyroid hormone-related protein and calcium levels after surgery and a rise in both before clinical relapse. However, the clinical relapse was itself associated with a fall in serum parathyroid hormone-related protein, nephrogenous cAMP and calcium, suggesting that the tumour had stopped producing parathyroid hormone-related protein or perhaps that post-translational processing had occurred as the tumour advanced. The tumour was investigated for parathyroid hormone-related protein mRNA content using reverse transcriptase polymerase chain reaction, both at diagnosis in surgically removed material, and using post-mortem specimens. The level of parathyroid hormone-related protein mRNA, while present, was much reduced in the recurrent tumour suggesting that active parathyroid hormone-related protein production fell substantially as the tumour advanced. This case suggests that, although demonstration of parathyroid hormone-related protein in hypercalcaemia is useful for diagnosis, tumoral secretion of this product may alter.