Polycomb (Pc) is involved in the stable and heritable repression of homeotic gene activity during Drosophila development. Here, we report the identification of a novel human Pc homolog, hPc2. This gene is more closely related to a Xenopus Pc homolog, XPc, than to a previously described human Pc homolog, CBX2 (hPc1). However, the hPc2 and CBX2/hPc1 proteins colocalize in interphase nuclei of human U-2 OS osteosarcoma cells, suggesting that the proteins are part of a common protein complex. To study the functions of the novel human Pc homolog, we generated a mutant protein, ⌬hPc2, which lacks an evolutionarily conserved C-terminal domain. This C-terminal domain is important for hPc2 function, since the ⌬hPc2 mutant protein which lacks the C-terminal domain is unable to repress gene activity. Expression of the ⌬hPc2 protein, but not of the wild-type hPc2 protein, results in cellular transformation of mammalian cell lines as judged by phenotypic changes, altered marker gene expression, and anchorage-independent growth. Specifically in ⌬hPc2-transformed cells, the expression of the c-myc proto-oncogene is strongly enhanced and serum deprivation results in apoptosis. In contrast, overexpression of the wild-type hPc2 protein results in decreased c-myc expression. Our data suggest that hPc2 is a repressor of proto-oncogene activity and that interference with hPc2 function can lead to derepression of proto-oncogene transcription and subsequently to cellular transformation.
The Polycomb group genes in Drosophila are involved in the stable and inheritable repression of gene expression. The Polycomb group proteins probably operate as multimeric complexes that bind to chromatin. To investigate molecular mechanisms of stable repression of gene activity in vertebrates we have begun to study Xenopus homologs of Polycomb group genes. We identified the Xenopus homologs of the Drosophila Polycomb gene and the bmi-1 gene. bmi-1 is a proto-oncogene which has sequence homology with the Polycomb group gene Posterior Sex Combs. We show that the XPolycomb and Xbmi-1 genes are expressed in overlapping patterns in the central nervous system of Xenopus embryos. However, XPolycomb is also expressed in the somites, whereas Xbmi-1 is not. We further demonstrate that the XPolycomb and Xbmi-1 proteins are able to interact with each other via conserved sequence motifs. These data suggest that also vertebrate Polycomb group proteins form multimeric complexes.
The insulin‐like growth factors I and II (IGF‐I and ‐II) are polypeptides which play an important role in growth and development of the organism. In the present report we describe the detection of human IGF‐I RNAs (both type Ia and type Ib) and IGF‐II RNAs in benign (leiomyoma) and malignant (leiomyosarcoma) tumours from smooth muscle origin, using Northern blot hybridization analysis. In normal smooth muscle tissue of the uterus we found low levels of IGF‐I RNAs only. In the tumours the same IGF‐I RNA species were detected as in adult non‐tumour tissues (uterus, liver). For transcription of the IGF‐II gene in these tumours, two promoters are used which are expressed in fetal liver, but not in adult liver. The presence of IGF‐I and IGF‐II RNAs was also established by nucleotide sequence analysis of recombinant DNA clones isolated from cDNA libraries derived from two leiomyosarcomas. The nucleotide sequences of these cDNA clones, together covering the entire coding regions of IGF‐Ia and IGF‐II var RNA, predict that IGFs encoded by the tumour RNAs do not differ in amino acid sequence from the corresponding polypeptides isolated from serum. In those tissues containing IGF‐I RNAs, IGF‐I immunoreactivity was also demonstrated.
A method based on the reverse transcriptase-polymerase chain reaction (RT-PCR) was developed that allows the determination of relative mRNA expression levels in fine-needle aspirates from human tumors. The method was developed for the c-erbB-2 gene, using the porphobilinogen deaminase (PBGD) gene as an internal standard. It was validated for mRNA isolated from cell lines and for material obtained by fine-needle aspiration from human breast cancer. Gene expression levels were determined by measuring the activity of radiolabeled RT-PCR-amplified gene-specific bands with a phosphor imager. At least four points are measured on the log-linear part of the amplification cycle versus signal intensity curves, and subsequently the distance between the curves of the gene of interest and that of an internal standard gene is used to calculate the relative expression levels. The method worked equally well with the BRCA1 gene, illustrating that it can be generalized to other genes. The method is suitable to measure or monitor semiquantitively gene expression levels in accessible human tumors in situ.
Recently, we presented evidence that protein kinase C (PKC) is involved in mediating the endogenous signals that induced competent Xenopus ectoderm to differentiate to neural tissue. We report here that PKC is already strongly activated in neural-induced ectoderm from midgastrula embryos and that this activation runs parallel with an increase in the level of inositol phosphates. We further identify several proteins that are phosphorylated, both in natural neural-induced ectoderm and in TPA-treated ectoderm, suggesting that they are phosphorylated through the PKC route. We found no major changes in PKC activity among different pregastrula stages, including the unfertilized egg. However, PKC isolated from animal, ectodermal cells is highly sensitive to Ca2+ and can be activated by low concentrations, (6–25 microM) of arachidonic acid, while PKC isolated from vegetal, endodermal cells is more insensitive to Ca2+ and cannot be activated by arachidonic acid. These results suggest that different PKC isozymes are present in animal and vegetal cells.
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