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Background/aims: Studies have identified a 67-kilodalton high-affinity laminin receptor (LR) whose expression has also been related to development, differentiation, and neoplastic transformation. The relationship of the 67-kilodalton LR to hepatic and enterocyte development and to enterocyte differentiation was investigated., Methods: LR messenger RNA (mRNA) was identified using a complementary DNA isolated from a rat crypt cell library. LR and integrin (alpha 6, beta 1, and beta 4) expression by rat intestinal crypt cells was compared with that of the more differentiated villus cells using Northern blotting. Developmental differences in LR expression were studied in fetal and neonatal rats. The pattern of LR expression in fetal and adult rat intestines was examined further by in situ hybridization., Results: LR mRNA levels were highest in fetal liver and intestine and adult rat crypt cells. LR mRNA levels were 9-10 times greater in crypt than in villus cells. Integrin subunit expression differed little between crypt and villus cells. Nascent transcription studies showed that the proportion of newly transcribed LR mRNA per total RNA synthesized was similar for crypt and villus cells, suggesting posttranscriptional control of LR mRNA levels in villus cells., Conclusions: Increased LR mRNA expression is a feature of the fetal intestine and of the undifferentiated, mitotically active crypt cells.

Cells from the Walker 256 carcinosarcoma, a rat breast tumor with a propensity to metastasize to bone, were labeled with [131I]5-iodo-2'-deoxyuridine and then added to 96-hour organ cultures of fetal Sprague-Dawley rat calvaria that had been prelabeled with 45Ca and incubated with various stimulators or inhibitors of resorption. In conditioned media from resorbing bone cultures, the number of cells that attached to the bone surfaces correlated with the degree of bone resorption (r = 0.65; P less than .005). The attachment response was maximal after 180 minutes of cocultivation and was inhibited by preincubation of the tumor cells with 10(-5) M cytochalasin B. Cellular attachment appeared to be promoted by a trypsin-sensitive factor released into the organ culture medium from resorbing bones. Enhanced tumor cell attachment did not appear to be related to a change in the surface properties of the resorbing bone, since it was not observed when the conditioned media were replaced with fresh medium. Furthermore, tumor cells placed in conditioned medium demonstrated increased attachment to plastic surfaces and formed aggregates. While there was a direct correlation between the ability of conditioned medium to promote cellular adhesion and chemotactic migration (r = 0.85; P less than .05), the factors responsible for chemotaxis and adhesion could be separated by gel filtration. The release of such factors from resorbing bones may promote the formation of secondary bone tumors, since in this system attachment of unlabeled cells was followed by proliferation of tumor cells and evidence of bone invasion.

Demineralized extracts of bone matrix and conditioned media from cultured fetal rat calvaria have been reported to contain growth stimulatory activity for bone cells. To investigate the potential role of these local bone growth factors in the development of bone metastases, we chose the Walker 256 carcinosarcoma, a rat mammary tumor which causes osteolytic bone metastases and hypercalcemia. 45Ca-labeled, 19-day fetal Sprague-Dawley rat calvaria were cultured for 96 hours in BGJb medium. Walker cells from ascites tumors or cultures were grown in unconditioned media or in conditioned media harvested from the bone cultures, in the presence of 10% fetal calf serum. Media were changed every 2 days, cells were counted daily for 5 days, and 3H-thymidine uptake into acid insoluble residues was measured. The growth of tumor cells was 5-6-fold greater in conditioned media than in unconditioned media and the effect was dose dependent. Cells cultured in conditioned media demonstrated a approximately 3-fold enhancement of 3H-thymidine incorporation. Generation of growth stimulatory activity correlated with the extent of bone resorption, measured by release of 45Ca from the fetal parietal bones (r = 0.85; P less than 0.001). Conditioned media from bones cultured with 10(-7) M prostaglandin E2 (PGE2) contained greater amounts of growth stimulatory activity than untreated conditioned media, but PGE2 itself did not stimulate tumor cell growth. Addition of 3.5 mM PO4 to bone cultures blocked bone resorption and the generation of growth factors. Growth stimulatory activity was stable to heat (56 C for 30 minutes) and trypsin digestion, with an apparent molecular weight of less than 17,000 daltons by high-performance liquid chromatography. Conditioned medium also stimulated the growth of 13762 rat mammary adenocarcinoma cells, MB-MDA-231 human breast carcinoma cells, TE-85 osteosarcoma cells, a murine fibrosarcoma and rat embryonic fibroblasts, with the most potent effects noted for Walker tumor cells, the TE-85 osteosarcoma, and human breast carcinoma lines. These results suggest a mechanism by which bone resorption could promote the development of skeletal metastasis.