Insulin-like growth factor-binding protein-5 inhibits growth and induces differentiation of mouse osteosarcoma cells.

The precise role of insulin-like growth factor-binding protein-5 (IGFBP-5) in regulating the growth of tumor cells, especially of bone-derived malignant cells, is not well understood. We have investigated the biological activity of IGFBP-5 by transfecting OS/50-K8 mouse osteosarcoma cells with an expression vector containing the osteocalcin promoter and the complete mouse IGFBP-5 cDNA (OC-IGFBP-5). Overexpression of IGFBP-5 mRNA and secretion of increased amounts of bioactive protein in conditioned media were demonstrated in different clones. For the analysis of cell proliferation, three clones exhibiting high levels of IGFBP-5 expression were selected and compared to a mock clone and to nontransfected parental cells. IGFBP-5-secreting clones displayed reduced proliferation under both anchorage-dependent and -independent conditions (P < 0.05). The increase in proliferation observed in IGFBP-5-secreting clones after addition of exogenous IGF was significantly lower than that observed in mock-transfected or parental cells. A similar result was obtained with long[R3]IGF-I which has a low affinity for all IGFBPs, suggesting that the inhibitory effect of IGFBP-5 is only partially IGF-dependent. OC-IGFBP-5-transfected clones expressed significantly higher amounts of osteocalcin mRNA (P < 0.05) and secreted more osteocalcin protein than a mock clone or parental OS-50/K8 cells. Thus, part of the growth-inhibiting effect of IGFBP-5 may be due to an induction of differentiation in these cells.

Overexpression of insulin-like growth factor-II in mouse embryonic stem cells promotes myogenic differentiation.

Embryonic stem (ES) cells derived from androgenetic or parthenogenetic mouse embryos are important tools for studying the roles of imprinted genes in early development. Androgenetic ES cells have been shown to preferentially differentiate into the myogenic lineage both in vitro and after formation of teratocarcinomas in vivo. To clarify if the maternally imprinted Igf2 gene which is expected to be overexpressed in androgenetic ES cells is sufficient to induce myogenic differentiation, R1 ES cells were transfected with human IGF-II expression vectors. Stable ES cell clones exhibiting human IGF-II mRNA and protein expression were studied vs ES cell clones without IGF-II overexpression in a standard in vitro differentiation system involving culture in "hanging drops" and observation of differentiation of the recovered embryoid bodies (EBs). EBs derived from IGF-II overexpressing ES cells showed stimulated myogenic differentiation evident by the appearance of myoblasts already 3 days after plating and by higher levels of skeletal muscle-specific transcripts (myf5, myoD, myogenin) at earlier stages. Our study demonstrates for the first time that overexpression of IGF-II enhances and accelerates myogenic differentiation of ES cells, which has implications for ES cell-derived tissue engineering.

Enzymatic activity of two caspases related to interleukin-1beta-converting enzyme.

Interleukin-1beta-converting enzyme is a member of a family of human cysteine proteases with specificity for aspartic acid, which have been named caspases. Within this family of enzymes, transcript X (TX) and transcript Y (TY) (caspases 4 and 5, respectively) are very similar to ICE (caspase 1) and form the ICE subfamily. Given the high degree of conservation in the sequences of these proteases (more than 50% amino acid identity in the mature enzymes), it was of interest to examine whether they shared similar substrate specificities. The three enzymes, ICE, TX and TY, were therefore expressed in baculovirus-infected insect cells, as 30-kDa proteins lacking the propeptide. Automaturation into p20 and p10 subunits occurred within the cells. Active ICE, TX and TY were collected in the cell culture supernatants. In addition, their production induced the activation of an endogenous 32-kDa putative cysteine protease (CPP32) like caspase. T7-tagged ICE, TX and TY were purified by immunoaffinity and tested for their catalytic efficiency on YVAD-containing synthetic substrates and on the ICE natural substrate, pro-interleukin-1beta. TX cleaved the same synthetic substrates as ICE (Km of 90 microM and k(cat) of 0.4 s(-1) for Suc-YVAD-NH-Mec, where Suc represents succinyl and NH-Mec represents amino-4-methylcoumarin) and could cleave pro-interleukin-1beta into the same peptides as ICE but less efficiently. On the other hand, TY showed very little efficacy on the different ICE substrates (Km of 860 microM for Suc-YVAD-NH-Mec). These results show that the ICE/TX/TY subfamily has functional heterogeneity and that ICE remains the preferred enzyme for pro-interleukin-1beta cleavage.

The DNA damaging drug cyproterone acetate causes gene mutations and induces glutathione-S-transferase P in the liver of female Big Blue transgenic F344 rats.

The gestagenic and antiandrogenic drug cyproterone acetate (CPA) is mitogenic, tumorigenic and induces DNA-adducts and DNA-repair synthesis in rat liver. Thus CPA is expected to be mutagenic. However in vitro mutagenicity test systems were negative. To examine whether CPA induces mutations in rat liver, the in vivo mutation assay based on Big Blue transgenic F344 rats was employed. Single oral doses of 25, 50, 75, 100 and 200 mg CPA/kg b.w. respectively were administered to female Big Blue rats. Six weeks after treatment, liver DNA was assayed for mutations. At the highest dose, 200 mg CPA/kg b.w., the frequency of (17 +/- 4) x 10(-6) spontaneous mutations was increased to a maximum of (80 +/- 8) x 10(-6) mutations. One-hundred and 75 mg CPA/kg b.w. resulted in mutation frequencies of (35 +/- 5) and (27 +/- 5) x 10(-6), respectively. The mutation frequency at doses of 50 and 25 mg CPA/kg b.w. was similar to that of vehicle treated controls. Statistical analysis of the dose-effect relationship revealed that it was not possible to decide whether a threshold dose exists or not. DNA adducts were analyzed by the 32P-postlabelling technique. The total level of the major and the two minor adducts observed in the autoradiograms increased between doses of 25 to 75 mg CPA/kg b.w. to a maximum of approximately 12,000 +/- 3000 adducts per 10(9) nucleotides. The level did not further increase significantly with 100 and 200 mg CPA/kg b.w. After CPA treatment no preneoplastic liver foci were observed. However, single glutathione-S-transferase placental form (GST-P) positive hepatocytes were observed and the frequency was dependent on the dose. These cells are not supposed to represent initiated cells, since they occurred only transiently after 6 weeks and disappeared thereafter completely. In conclusion, our results demonstrate that CPA is mutagenic in vivo. The mutation frequency increased at high CPA doses, when the increase of the DNA adduct formation had already ceased. This suggests that the mitogenic activity of CPA is required to express the mutations.

Somatic and germ cell mutagenesis in lambda lacZ transgenic mice treated with acrylamide or ethylnitrosourea.

The transgenic Muta Mouse in vivo mutagenesis assay was employed to determine the activity of acrylamide and ethylnitrosourea in liver and germ cells after 3, 10 and 100 days following treatment. Each cell of the Muta Mouse carries 80 copies of the lambda gt10 phage including the bacterial lacZ gene, which act as the target gene for the mutagenesis assay. Groups of Muta Mice were given a single intraperitoneal injection of 80 or 160 mg/kg ethylnitrosourea or 50 or 100 mg/kg acrylamide. The tissues were prepared 3, 10 or 100 days post treatment. The liver genomic DNA was extracted with the manufacturer's standard protocol, while the genomic germ cell DNA was extracted with 4 different methods due to problems encountered in DNA yields and packaging efficiency. The mutation analysis of the lacZ gene was carried out by the positive selective assay method [Gossen et al. (1989) Proc. Natl. Acad. Sci. USA, 86, 7971-7975; Dean and Myhr (1994) Mutagenesis, 9, 183-185]. There was a slight increase due to treatment of the observed mutation frequencies in the acrylamide liver group for all three assay times. From the day 3 group to the day 100 group a time dependent decrease in all the absolute mutant frequencies was detectable. The ethylnitrosourea liver group showed a time- and dose-dependent increase in the mutant frequencies from day 3 to day 100. No meaningful results were obtained for the germ cell tissue assays due to the low amount of genomic DNA extracted which was not packageable in the lambda lacZ assay. At present for the mutagenesis assay of isolated spermatozoa in our laboratory we would be forced to pool tissues from animals to obtain enough DNA for an assay. Since 'jackpot'-animals may exist [Heddle et al. (1992) Mutation Res., 272, 195-203] the individual animals of such a pooled analysis group must be tested before pooling.

Amino acid changes outside the G-H loop of capsid protein VP1 of type O foot-and-mouth disease virus confer resistance to neutralization by antipeptide G-H serum.

Antiserum to a peptide corresponding to the 135-154 sequence of capsid protein VP1 of the foot-and-mouth disease virus O1 Kaufbeuren was raised in a pig. Although this serum contained neutralizing antibodies, the pig showed clinical symptoms after challenge. Virus isolated from this pig was identified as a mutant, with changes at positions 50, 198 and 211 of VP1 and at position 209 of VP2. This mutant, as well as a plaque isolate of it, differing from the challenge virus at positions 198 on VP1 (alanine being substituted for glutamic acid) and 209 on VP2 (histidine being substituted for tyrosine) resisted neutralization by the anti-peptide serum also in vitro. The same was observed with the O1 Kaufbeuren-related strain O1 Burgwedel, isolated from cattle in the field. It had substitutions only at positions 43 and 101 on VP1. The results show that neutralization epitopes flanking positions 145-147 on VP1 are modulated by other capsid protein parts. These parts seem to be important for neutralization escape in natural FMDV host species.