The primary structure of human hemopexin deduced from cDNA sequence: evidence for internal, repeating homology.

We have cloned and analyzed a cDNA containing the coding sequence for human hemopexin. We have first identified, by immunological screening of 30.000 colonies of a liver cDNA library in the expression vector pEX1, a clone carrying an insert 1170 base pairs long that shows 100% homology with a known human hemopexin peptide. The complete sequence coding for hemopexin was isolated from a liver cDNA library in the vector pAT218. The DNA insert of 1523 base pairs shows an open reading frame coding for 439 amino acids, a 3' noncoding region of 159 nucleotides long, followed by a poly(A) tail. The insert spans the entire coding region and from which the primary structure of the protein was deduced. By computer assisted analysis of the amino acid sequence, it was possible to recognize a core unit, of about 45 amino acids, which is repeated 8 or possibly even 10 fold along the polypeptide chain. This feature suggests that the gene might have evolved through a series of duplications. This characteristic, together with prediction of secondary structure, suggest a rough model for the tridimensional folding that allows some speculations on the function of hemopexin. Blot hybridization of total RNA from human liver with nick translated hemopexin cDNA detected a message of about 1600 nucleotides. Southern blot experiments to identify the hemopexin gene (s) suggest that it is not a large multi-gene family, but that there is only one or at most a few genes in the human genome.     

Isolation and characterization of the major form of human MUC18 cDNA gene and correlation of MUC18 over-expression in prostate cancer cell lines and tissues with malignant progression.

Ectopical expression of huMUC18, a cell adhesion molecule in the immunoglobulin gene superfamily, causes a non-metastatic human melanoma cell line to become metastatic in a nude mouse system. To determine if MUC18 expression correlates with the development and malignant progression of prostate cancer, we investigated differential expression of human MUC18 (huMUC18) in normal prostate epithelial cells, prostate cancer cell lines, and prostatic normal and cancer tissues. We cloned and characterized the human MUC18 (huMUC18) cDNA gene from three human prostate cancer cell lines and three human melanoma cell lines. The cDNA sequences from the six human cancer cell lines were identical except differences in one to five nucleotides. The deduced amino acid sequences of the longest ORF were 646 amino acids that were identical in these cDNAs except for one to three amino acid residues. The amino acid sequences of all our huMUC18 cDNA genes are similar to that cloned by other group (GenBank access #M28882) except differences in the same seven amino acids. We conclude that huMUC18 cDNA gene reported here represents the gene product from a major allele. The MUC18 mRNA and protein was expressed in three metastatic prostate cancer cell lines (TSU-PR1, DU145, and PC-3), but not in one non-metastatic prostate cancer cell line (LNCaP.FGC). The expression of huMUC18 in these four cell lines is positively related to their extent of in vitro motility and invasiveness and in vivo metastasis in nude mice. HuMUC18 protein was also expressed at high levels in extracts prepared from tissue sample sections containing high grade prostatic intraepithelial neoplasia (PIN), but weakly expressed in extracts prepared from cultured primary normal prostatic epithelial cells and the normal prostate gland. Immunohistochemical analysis showed that huMUC18 was expressed at higher levels in the epithelial cells of high-grade PIN and prostatic carcinomas, and in cells of a perineural invasion, a lymph node, and a lung metastases compared to that in normal or benign hyperplastic epithelium (BPH). We therefore conclude that MUC18 expression is increased during prostate cancer initiation (high grade PIN) and progression to carcinoma, and in metastatic cell lines and metastatic carcinoma. Increased expression of MUC18 is implicated to play an important role in developing and malignant progression of human prostate cancer. Furthermore, the lacking of predominant cytoplasmic membrane expression of MUC18 appeared to correlate with malignant progression of prostate cancer.     

Expression of wild-type p53 is not compatible with continued growth of p53-negative tumor cells.

Inactivation of the cellular p53 gene is a common feature of Friend virus-induced murine erythroleukemia cell lines and may represent a necessary step in the progression of this disease. As well, frequent loss or mutation of p53 alleles in diverse human tumors is consistent with the view of p53 as a tumor suppressor gene. To examine the significance of p53 gene inactivation in tumorigenesis, we have attempted to express transfected wild-type p53 in three p53-negative tumor cell lines: murine DP16-1 Friend erythroleukemia cells, human K562 cells, and SKOV-3 cells. We found that aberrant p53 proteins, which differ from wild-type p53 by a single amino acid substitution, were expressed stably in these cells, whereas wild-type p53 expression was not tolerated. The inability of p53-negative tumor cell lines to support long-term expression of wild-type p53 protein is consistent with the view that p53 is a tumor suppressor gene.     

杆状病毒对不同哺乳动物细胞基因转移及表达效率的研究

研究已证实杆状病毒可进入某些哺乳动物细胞,这提示了可将重组杆状病毒作为一种对哺乳动物细胞的新型基因转移载体。利用已构建的重组杆状病毒BacV-CMV-EGFPA,以含病毒的Sf9细胞培养上清同时孵育多种哺乳动物细胞,利用流式细胞术检测报告基因在不同细胞株中的转移效率及表达效率。共使用了20种哺乳动物细胞株,其中有12种人类组织细胞,7种小鼠组织细胞,1种猴组织细胞。实验结果显示携带CMV启动子的重组杆状病毒可有效进入多数哺乳动物细胞,其中对人、猴来源细胞的基因转移效率显著高于对鼠源细胞,对贴壁细胞的基因转移效率显著高于对悬浮细胞。同时,通过脂质体LipofectAMINE将携带有CMV启动子和EGFP基因的哺乳动物细胞表达质粒pCDNA3-1-EGFP分别转染部分特别是被认为杆状病毒进入能力较低的细胞株,结果显示CMV启动子在这些细胞中均可有效引导EGFP基因的表达,因此认为携带了CMV启动子的重组杆状病毒对不同哺乳动物细胞基因转移效率能基本反映出杆状病毒对不同种哺乳动物细胞的进入能力。通过综合比较携带CMV启动子的杆状病毒对不同种属及组织来源细胞的基因转移及表达效率,可看出杆状病毒对灵长类动物贴壁细胞的基因转移及表达效果是较好的,而对小鼠来源的细胞及悬浮培养细胞却并不十分理想,这表明将重组杆状病毒作为一种对哺乳动物细胞的基因转移工具,仍有其局限性,不一定对所有的细胞都合适,在应用时应予以充分考虑。     

A novel human hepatoma cell line, FLC-4, exhibits highly enhanced liver differentiation functions through the three-dimensional cell shape

We characterized three-dimensional human hepatoma cell lines, functional liver cell (FLC) cell lines, to establish a highly differentiated hepatoma cell line. We investigated the effect of extracellular matrix and cell morphology on liver-specific gene expression in FLC cells. The hepatocyte nuclear factor-4α (HNF-4α) and other liver-specific gene expressions were enhanced in spherical FLC-4 cells on EHS-gel, but other human hepatoma cells such as HepG2 did not show the enhancement. Importantly, the liver-specific gene expression levels in spherical FLC-4 cells cultured on EHS-gel were comparable to those of human liver and were much higher than those of other human hepatoma cell lines. The major matrix components and growth factors in EHS-gel did not affect cell shape and liver functions. To exclude any effect of the extracellular matrix, we made spherical FLC-4 cells by actin filament disruption. The actin-disrupted spherical cells also showed an enhanced liver-specific gene expression. We concluded that three-dimensional cell shape per se is one of the most important determinants of liver differentiation functions in FLC-4 cells. Cell morphology-dependent induction of liver-specific gene expression was mediated through microtubule organization. In conclusion, differentiation of FLC-4 human hepatoma cell line can be enhanced to a human liver-like level through the three-dimensional cell shape in a microtubule-dependent manner.