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Proteins in porcine amniotic fluid and sera (both fetal and adult) were separated electrophoretically in sodium dodecyl sulfate-containing polyacrylamide gels and transferred to nitrocellulose sheets. Western blots were analysed for proteins that would bind (a) radioiodinated insulin-like growth factor-I (IGF-I) and (b) antibodies to a rat insulin-like growth factor binding protein. Multiple insulin-like growth factor binding proteins were identified in sera. The binding proteins ranged in size from Mr 192,000 to 26,000. One immunologically cross-reactive protein (Mr 36,000) was detected. No binding proteins were detected routinely in amniotic fluids. Sera from adult swine were fractionated by preparative isoelectric focusing. Two binding proteins (Mr 192,000, 46,000) were located in acidic fractions which also contained IGF-I and IGF-II. Two binding proteins (Mr 36,000, 26,000) were located in neutral to basic fractions which contained primarily IGF-II. Immunoglobulin-sized material from adult sera fractionated over Sephadex G-200 contained two binding proteins (Mr 46,000, 42,000) whereas albumin-sized material contained one (Mr 36,000). Porcine insulin-like growth factor binding proteins are as heterogeneous as those from humans.

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Previous studies have shown that the binding of fibroblast growth factor to several different non-transformed cell lines decreases as cell density increases. However, it was not determined whether this is due to a reduction in receptor number or to a decrease in receptor affinity. In this study, we demonstrate that the reduction in fibroblast growth factor binding is due to a reduction in receptor number. In addition, flow cytometric analysis indicated little or no difference in the cell cycle distribution of the cells at low and high cell densities, yet the binding of fibroblast growth factor was reduced substantially at high cell densities. Thus, the reduction in growth factor binding observed in this study does not appear to result from cell cycle-dependent regulation of growth factor receptors.

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The interferons (IFNs) have been shown to be antagonistic to the growth stimulatory effects of mitogens on cultured cells. A report of the interactions of IFN-beta and platelet-derived growth factor on BALB/c-3T3 mouse cells established that IFN itself induced the secretion of a limited number of proteins from this cell line. The present work was undertaken to determine if other murine cell lines treated with homologous IFN-beta also secreted new or additional protein(s) in response to this agent and if this response correlated with other phenotypic properties of the cells. The cell lines examined included L929 cells and two derivatives of this line (GM347 and WDIFN), CAK-TK-, Swiss-3T3, and BALB/c-3T3. Each line was exposed to [35S]methionine in the absence and in the presence of IFN-beta, the supernatant fluids collected, and the radioactive, secreted proteins examined by fluorography after electrophoresis through SDS-containing polyacrylamide gels. Two cell lines (GM347 and Swiss-3T3) did not appear to secrete new or additional proteins after IFN treatment. However, four lines (L929, WDIFN, CAK-TK-, and BALB/c-3T3) did secrete new or additional proteins in response to IFN. Thus IFN-induced secretion of protein appeared to be a common but not universal phenomenon. In addition, although the number and apparent size(s) of the IFN-induced, secreted proteins were different in these various lines, one protein (Mr = 89-90,000) appeared to be secreted by each of them. In this respect it was unique. Moreover the IFN-induced secretion of protein did not appear to correlate with the antiviral or antiproliferative effects of IFN.

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The interferon (IFN)-dependent induction of two double-stranded RNA-dependent enzymes was examined in L cells and an L-cell variant (WDIFN) which is highly resistant to the inhibitory effects of IFN on cellular multiplication. IFN, in a concentration-dependent manner, inhibited the multiplication of parental L cells and induced increased levels of the double-stranded RNA-dependent enzymes in parental L cells. Although WDIFN cells were resistant to the antiproliferative effects of IFN, the cells responded to IFN by increasing their levels of the double-stranded RNA-dependent enzymes. However, the level of activity of each enzyme was lower in the WDIFN line than the parental line when both lines were treated with similar concentrations of IFN. The reduced response of the WDIFN line was not the result of the line being a heterogeneous population of cells nor of IFN being more unstable in the presence of WDIFN cells. In addition there was no evidence that WDIFN cells produced a mitogenic factor that could overcome the antiproliferative effects of IFN, nor that sodium butyrate could increase the sensitivity of WDIFN cells to the antiproliferative effects of IFN.

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L cells grown for 18 to 22 months in the presence of interferon (IFN) retained their sensitivity to the antiviral effect of IFN but were less sensitive to cell growth inhibition by IFN. Moreover, the parental and variant lines became hyporesponsive (i.e. less responsive to virus induction of IFN) with different kinetics after IFN treatment. Experiments using mitomycin C to inhibit cell division suggested that cell division is required to enter the hyporesponsive state.

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Pre-treatment of L cells with 750 units/ml of mouse interferon depressed the yield of interferon when the cells were treated with Newcastle disease virus (NDV). The refractory state persisted for more than 5 days. Nucleated fragments (karyoplasts) obtained by cytochalasin B-induced enucleation were made from such interferon pre-treated cells; cells reconstituted from these karyoplasts produced less interferon in response to NDV than did cells reconstituted from karyoplasts derived from untreated cells. The source of the enucleated cytoplasm (cytoplasts) used in the cellular reconstitutions did not affect the yields of interferon.

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We have obtained hybrids of PCC4-aza 1, a mouse embryonal carcinoma stem cell line, and two different thymidine kinase deficient mouse cell lines. We have examined the ability of the parental and hybrid cells to produce interferon after infection with the Newcastle Disease virus and to enter the antiviral state when treated with mouse interferon. The interferon system of PCC4-aza 1 is inactive; this characteristic is recessive in the hybrids obtained.

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The expression of the interferon-induced antiviral state was studied in heterokaryons and cytoplasmic hybrids (cybrids). An autoradiographic assay for the antiviral state, in which the percentage of cells containing vaccinia viral DNA factories was determined, was used. The expression of the antiviral state was dominant in homokaryons and heterokaryons formed by fusion of interferon-treated cells with untreated cells. Cytoplasts derived from treated cells conferred resistance to virus growth on cybrids formed by fusing such cytoplasts with untreated cells. Treatment of L cell x HeLa cell heterokaryons with human interferon or mouse interferon was much less effective in inducing a detectable antiviral state than was similar treatment of parental cells with homospecific interferon. The antiviral state was fully induced when heterokaryons were treated simultaneously with both types of interferon. Cybrids formed by fusing L cell cytoplasts with HeLa cells or HeLa cytoplasts with L cells did not enter a detectable antiviral state after treatment with interferon specific for the cell type of the enucleated parent. However, treatment of cybrids with interferon specific for the cell type of the nucleated parent was effective in inducing a detectable antiviral state.

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Enucleation of L cells leads to loss of the capacity to produce interferon, showing that the cell nucleus is essential for interferon formation. However, when the cells were enucleated while interferon formation. However, when the cells were enucleated while interferon formation was proceeding, the cytoplasts were capable of continuing to synthesize interferon by a process shown to be protein synthesis, showing that the interferon messenger RNA leaves the nucleus after synthesis. Reconstructed cells were obtained by Sendai virus fusion of karyoplasts and cytoplasts. Such reconstructed cells were capable of producing at least as much interferon (43 interferon units/10(4) nucleated cells) as control cells (31 interferon units/10(4) nucleated cells).

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Although DNA polymerase-alpha (DNA nucleotidyltransferase; deoxynucleoside triphosphate: DNA deoxynucleotidyltransferase; EC 2.7.7.7) probably functions in the nucleus, it is usually found predominantly in the nonnuclear fraction of disrupted cells. We have reexamined the intracellular location of this enzyme using cytochalasin-B-induced enucleation, a technique which avoids exposure of nuclei to extra-cellular conditions during cell fractionation. In conditions where viability of separated cell parts is high and recovery is quantitative, we find greater than 85% of total DNA polymerase-alpha (and DNA polymerase-beta) activity in the nucleated cell fragments (karyoplasts), from which we conclude that the location in vivo of DNA polymerase-alpha is either nuclear or perinuclear. On the other hand, thymidine kinase (ATP: thymidine 5'-phosphotransferase, EC 5.7.1.75) is found primarily in the enucleated cell fragments (cytoplasts). The enucleation procedure used in this work should be of general use for intracellular location studies.

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Mouse L929 cells were separated into enucleated cytoplasmic components (cytoplasts) and nucleated subcellular fractions (karyoplasts) in the presence of cytochalasin B. Karyoplasts from cells containing tritiated nuclei were fused, using inactivated Sendai virus, to cytoplasts from cells containing large (1.0-mum diameter) latex spheres in the cytoplasm. Mononucleated cells containing radioactive nuclei and large latex spheres in the cytoplasm were observed among the products of the fusion reaction. Some of these cells were in mitotic configurations. The results indicate that cells capable of undergoing mitosis can be reconstructed from the products of cellular enucleation in the presence of cytochalasin B.

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