Molecular cloning, expression, localization, and gene organization of PTX1, a human nuclear protein that is downregulated in prostate cancer.

A cDNA, designated PTX1, has been isolated by subtractive hybridization on the basis that it is expressed in normal prostate but not in prostate carcinoma. The full-length cDNA was subsequently established by 5' and 3' RACE. Nucleotide sequence analysis of the 5'- and 3'-RACE clones yielded a composite cDNA of 1327 bp, which predicted a protein of 377 amino acid residues with a putative nuclear import signal (RRLNRKK) at its N terminus. The PTX1 gene was localized to human chromosome 12 and was found to be ubiquitously expressed. A segment of the cDNA was expressed in E. coli to produce a fragment of the PTX1 protein for the generation of specific antibodies. The resulting antibodies detected a 73-kDa protein in both nuclear and cytoplasmic extracts of prostate, although the level in the cytoplasmic extract was much lower. Using immunohistochemical analysis, the PTX1 protein was localized mainly in the nuclei of glandular epithelia of normal prostate. The nuclear staining was greatly reduced in prostate carcinoma. The gene organization of PTX1 was established by comparing the cDNA sequence with the published human genomic sequence.     

Isolation and partial characterization of perichromatin granules: A unique class of nuclear RNP particles

Perichromatin granules (PCG) have been isolated from cycloheximide-treated rat liver nuclei by a procedure that preserved their ultrastructural characteristics. Like the PCG particles in situ, the isolated granules were 300–400 Å in diameter; they had an approximate sedimentation coefficient of 40S. The Bernhard bleaching procedure showed that the isolated perichromatin granules are not chromatinous components. A low molecular weight 4.7S RNA approx. 100 nucleotides long was associated with the granules. Analysis of the proteins of the isolated perichromatin granules on SDS polyacrylamide gel electrophoresis showed one major polypeptide (mol. wt approx. 34 000) along with two other minor polypeptides (mol. wt 31 000 and 38 000). The major polypeptide found in the perichromatin granules had similar migration characteristics on SDS gels to a peptide found in both rat liver and HeLa cell heteronuclear ribonucleoprotein (hnRNP) particles.     

Evidence from studies on segregated nucleoli that nucleolar silver staining proteins C23 and B23 are in the fibrillar component

Several procedures for the silver staining of nucleoli have been evaluated at the electron microscopic level to determine optimal conditions for ultrastructural preservation and staining specificity. The present study shows that a brief fixation with 1% buffered formaldehyde followed by methanol: acetic acid (3 : 1) fixation yielded optimal preservation and silver staining of nucleoli. Using this procedure for electron microscopic studies of interphase nucleoli, it was found that the punctate silver grains observed by light microscopy were composed of fine silver granules, of approx. 100 Å diameter, organized in discrete clusters. In similar studies on adriamycin-induced segregated nucleoli, it was observed that the silver staining reaction was mainly limited to the fibrillar portion of the nucleolus. Accordingly, nucleolar proteins C23 and B23, found earlier to be the major silver binding proteins of the nucleolus, are mainly concentrated in the fibrillar nucleolar component.     

Two-dimensional electrophoresis of proteins of citric acid nuclei prepared with aid of a Tissumizer

Nuclei prepared from normal rat liver and Novikoff hepatoma ascites cells with the aid of a Tissumizer® in media containing 0.5 and 5 % citric acid were compared on the basis of electron microscopic appearance, DNA, RNA and protein content. Electron microscopy revealed better preservation of the nucleolar and nuclear morphology in the nuclei isolated in 0.5 % citric acid than in nuclei isolated in 5 % citric acid. Moreover, losses of protein and DNA from liver nuclei prepared by the sucrose-Ca²⁺ procedure were significantly less in nuclei treated with 0.5 % citric acid than in nuclei treated with 5 % citric acid. The preservation of nuclear morphology and the retention of the majority of types of nuclear protein were significantly better with the procedure using 0.5 % citric acid than with the procedure using 5 % citric acid. The 5 % citric acid treatment was found to alter nuclear morphology and extract specific nuclear proteins, as demonstrated by two-dimensional polyacrylamide gel electrophoresis of the proteins.     

Influence of growth rate on protein synthesis and distribution of ribosomes in HeLa cells.

HeLa cells have been grown at different rates in steady-state continuous and semi-continuous culture. Slowly growing cells contain more protein and less RNA than rapidly growing cells, but appear to synthesize protein by less efficient use of the available RNA. The rate of RNA accumulation increases rapidly with increasing growth rate and rapidly growing cells contain more ribosomal subunits, and more and larger polysomes, but have fewer monoribosomes than slowly growing cells.     

PTX1(ERGIC2)-VP22 fusion protein upregulates interferon-beta in prostate cancer cell line PC-3

PTX1 is a gene identified by subtractive hybridization on the basis that it is expressed in normal prostate and not in prostate carcinoma. It is unrelated to the pituitary homeobox protein (Ptx1 or Pitx1), which regulates pituitary hormone gene expression, and its function is currently unknown. Recently, it was found to be a homolog of the yeast Erv41p, an endoplasmic reticulum (ER) resident protein involved in protein trafficking between ER and Golgi, and was renamed as ERGIC2. Ectopic expression of a partial sequence of PTX1 (Met84 - Leu225) as a VP22-fusion protein in prostate cancer cell line, PC-3, induced cellular senescence. Gene expression microarray analyses showed that interferon-beta (IFN-beta) and a number of IFN-inducible genes, among other genes, were upregulated by the PTX1-VP22 fusion protein. Upregulation of IFN-beta was confirmed by RTPCR and promoter-reporter assay. However, the upregulation of IFN-beta by the PTX1-VP22 fusion protein was not due to nuclear translocation of the PTX1 luminal domain.     

Effects of PTX1 expression on growth and tumorigenicity of the prostate cancer cell line PC-3

PTX1 is a gene identified by subtractive hybridization on the basis that it is expressed in normal prostate and not in prostate carcinoma. It encodes a nuclear protein that is downregulated in prostate carcinoma. Expression constructs containing PTX1 cDNA in both sense and antisense orientations were transfected into prostate tumor cell line, PC-3 cells. The effects of the expression of PTX1 and antisense PTX1 on PC-3 cells were examined using cell growth, proliferation, soft agar, invasion chamber, senescence-associated beta-galactosidase, and nude mice assays. Cells transfected with PTX1 construct in the sense orientation were growth-arrested. These cells displayed multiple morphological changes consistent with cellular senescence, including the expression of a senescence-associated beta-galactosidase. On the other hand, expression of antisense PTX1 RNA in PC-3 cells resulted in uncontrolled cell growth and increase of invasive potential. In nude mice, cells expressing antisense PTX1 grew sixfold faster than the control. These results suggest that PTX1 may play an important role in the growth and tumorigenicity of PC-3 cells.