Insulin-like growth factor-I induces cyclin-D1 expression in MG63 human osteosarcoma cells in vitro.

The insulin-like growth factors (IGFs) stimulate cell division by modulating events occurring during the prereplicative (G1) phase of the cell cycle, but identification of the critical events has proved difficult. Recent observations suggest that progression through the cell cycle is dependent on the activation of a group of serine-threonine-specific protein kinases whose activities are regulated by accessory proteins, termed cyclins. The identification of cyclin species expressed during G1 has led to the hypothesis that modulation of cyclin expression may be the critical event regulated by growth factors. The present studies were undertaken to determine whether the IGFs regulate the expression of specific G1 cyclins in MG63, a human cell line that is unusually responsive to IGF, and to characterize this effect. We found that in these cells IGF-I stimulates the cyclin-dependent kinases, and that stimulation is associated with an increase in cyclin-D1 mRNA and protein expression. The increase in cyclin-D1 occurs early in G1 and corresponds to the portion of the cell cycle in which IGF acts on these cells. The increase in cyclin-D1 mRNA is due at least in part to an increase in the rate of transcription initiation of the gene. The mRNA levels of cyclin-B1 (a G2 cyclin) and two cyclin-dependent kinases, cdc2 and cdk2, also increased in response to IGF, but at later times. These results are consistent with the hypothesis that IGF modulation of D-type cyclin expression plays a role in the regulation of cell replication.

Effects of insulin-like growth factor receptor inhibition on human melanomas in culture and in athymic mice.

The role of the insulin-like growth factor (IGF) receptor in regulating the growth of melanoma cells was evaluated by examining the effect of antibody-mediated IGF receptor inhibition on the growth of four human melanoma cell lines in culture and as xenotransplants in athymic mice. All four cell lines expressed typical type I IGF receptors and an antibody to this receptor (alpha IR-3) inhibited [125I]IGF-I binding. However, the cell lines varied widely in their in vitro responsiveness to IGF-I and alpha IR-3: in the WM 373 and WM 852 cell lines, IGF-I stimulated cell replication and alpha IR-3 inhibited this response, whereas in the WM 239-A and WM 266-4 cell lines neither the growth factor nor the antibody affected growth. A wide variation was also observed in the effect of the antibody on the growth of the different cell lines as xenotransplants but this qualitatively correlated with the responses observed in vitro: alpha IR-3 treatment significantly inhibited the growth of the WM 373 and WM 852 xenotransplants but did not inhibit the growth of the WM 239-A or WM 266-4 xenotransplants and may even have had a slight stimulatory effect. These results indicate that the IGF receptor pathway is a functional regulator of the in vivo growth of some melanomas and that this is reflected in the activity of this pathway as determined in vitro. These findings suggest that therapies aimed at inhibiting the IGF pathway may be beneficial in treating some melanomas.

Effect of IL-1 on experimental bone/bone-marrow metastases.

Bone metastasis is a common event and a major cause of morbidity in cancer patients. The hematopoietic marrow of the bones, rather than the bone tissue per se, is the target organ in bone metastasis. In the bone marrow, IL-1 induces the release of hematopoietic growth factors that may affect tumor-cell growth. We treated groups of mice with rhuIL-1 alpha to examine its role in the establishment of experimental bone/bone-marrow metastasis. We found that injection of 2 micrograms of rhuIL-1 alpha 24 hr prior to, simultaneously with or 24 hr after the injection of 10(4) B16 melanoma cells into the left cardiac ventricle of mice resulted in a 2-fold increase in the average number of colonized bones per mouse. GM-CSF is produced by bone-marrow stromal cells in response to IL-1, and its receptor has been found on tumor cells, including melanoma cells. However, the administration of rmuGM-CSF to mice by either multiple injections or continuous infusion did not affect the number of colonized bones. Many of the biologic effects of IL-1 are mediated by prostaglandins. Treatment of mice with 100 micrograms of indomethacin, a potent inhibitor of prostaglandin synthesis, prior to the injection of rhuIL-1 alpha, prevented the increase in number of bone metastases. To determine whether constitutive productions of IL-1 and/or prostaglandins are involved in the pathogenesis of bone/bone marrow metastasis, we treated mice with antimouse IL-1 alpha neutralizing antibodies, rhuIRAP (an inhibitor of IL-1 activity) or indomethacin. We found no difference in the average number of colonized bones per mouse between treated and control mice. We conclude that exogenous administration of IL-1 enhances experimental bone/bone-marrow metastases, and that this phenomenon is mediated through prostaglandins. However, neither the constitutive production of IL-1 nor that of prostaglandins appear to play a role in the pathogenesis of bone/bone-marrow metastasis in our murine model system.

Incidence and distribution of experimental metastases in mutant mice with defective organ microenvironments (genotypes Sl/Sld and W/Wv).

Mice carrying mutations at the Sl (steel) and W (dominant white spotting) loci develop abnormalities on 3 migratory embryonic stem cell populations: hematopoietic stem cells, neural crest-derived melanocytes, and primordial germ cells. Transplantation experiments have indicated that the Sl locus affects the microenvironment where stem cells migrate, proliferate, and differentiate, while the W locus affects the migratory cells themselves. The Sl locus encodes for a multipotent growth factor known as stem cell factor. The W locus encodes the c-kit protein tyrosine kinase receptor whose ligand is the stem cell factor. We have investigated the incidence and organ distribution of experimental metastases after systemic intra-arterial injection of B16-G3.26 melanoma cells into mutant Sl/Sld and W/Wv mice. Both mutant mouse strains had a markedly lower incidence of ovarian metastases when compared with their congenic +/+ mice. In contrast to the rare colonization of the ovaries, Sl/Sld and W/Wv mice developed metastases in the myocardium, kidney, and stomach--anatomic sites that were infrequently or never affected in their congenic nonmutant mice. The only organs in which the average number of metastatic colonies differed between Sl/Sld and W/Wv mice were the bone marrow and kidneys. The average number of colonized bones per mouse in the Sl/Sld group was 5.0 +/- 3.1 (SD), compared with 12.7 +/- 5.3 in the W/Wv group. The average number of metastatic nodules in the kidneys of Sl/Sld mice was 24.6 +/- 9, while W/Wv mice had 15.5 +/- 2.5. Mutant mice with multiple metastatic nodules in the kidneys, heart, and stomach were also found to have forestomach papillomas, an enlarged duodenum, kidney abnormalities, and small body size. The results of this study provide useful information on potential mechanisms of interaction of metastatic cells with their target organs, and suggest that there are additional organ defects associated with the mutations in the Sl and W loci. They also document the importance of mutant mice in metastasis research.