Kong-Bung Choo

A common precursor for a putative hemorrhagic protein and rhodostomin, a platelet aggregation inhibitor of the venom of Calloselasma rhodostoma: molecular cloning and sequence analysis.

Rhodostomin is a platelet aggregation inhibitor secreted by the venom gland of Calloselasma rhodostoma. We report here the isolation of a 1.67-kilobase (kb) lambda gt11 cDNA clone using degenerate oligonucleotide probe based on a partial amino acid sequence of rhodostomin. The amino acid sequence deduced from an open reading frame of the cDNA indicates that (i) the 68-amino acid sequence of rhodostomin is located at the carboxyl terminus of the precursor polypeptide and (ii) a peroxisomal targeting sequence (ser.his.ala.) exists between the stop codon and the rhodostomin sequence of the precursor. Since the amino-terminal segment of the deduced sequence shows a high degree of identity with hemorrhagic proteins, which are zinc-metalloproteinases, found in the venom of some crotalid and viperid snakes, our results also predict the existence of at least one such hemorrhagic protein in the venom of Calloselasma rhodostoma. The derivation of a platelet aggregation inhibitor and a hemorrhagic protein from the same precursor protein is consistent with the fact that these proteins may be synergistic in function.

Gender determination in single bovine blastomeres by polymerase chain reaction amplification of sex-specific polymorphic fragments in the amelogenin gene†

A sensitive technique for the sexing of bovine embryos was developed using polymerase chain reaction (PCR) amplification of the bovine amelogenin (bAML) gene on the X‐ and Y‐chromosomes of Holstein dairy cattle. Cloning and DNA sequencing showed a 45.1% homology between the fifth intron of the bAML‐X and bAML‐Y gene with multiple deletions. A pair of sex‐specific primers was designed to allow amplification of a single fragment of 467‐bp from the X‐chromosome of female cattle and two fragments of 467‐bp and 341‐bp from the X‐ and Y‐chromosomes of male cattle. The primers were successfully applied to bovine sexing from single blastomeres isolated from day‐6 to day‐7 cow embryos by direct cell lysis and PCR. Our protocol of embryo sexing should be applicable to the diagnosis of defective genes in vitro in human embryos and in other domestic or recreational animals. Mol. Reprod. Dev. 54:209–214, 1999.

Molecular Analysis of Cellular Loci Disrupted by Papillomavirus 16 Integration in Cervical Cancer: Frequent Viral Integration in Topologically Destabilized and Transcriptionally Active Chromosomal Regions

To discern the structural features of cellular loci that are disrupted by type 16 human papillomavirus (HPV‐16) integration in cervical cancer, a polymerase chain reaction (PCR)‐based strategy was employed for direct amplification and sequence analysis of four such cellular loci in cancer biopsy samples. One of the HPV‐16‐disrupted loci was found to be the microtubule‐associated protein (MAP‐2) gene and the other three loci were uncharacterized and were designated PID‐1 to ‐3 (for papillomavirus integration‐disrupted). The junctional sequences of the viral integration sites in the four loci analyzed are bracketed by long tracts of homogeneous purine or pyrimidine or alternating purine‐pyrimidine which are known to destabilize the B‐form conformation of the DNA structure. Using a panel of human/hamster hybrid cell DNAs and PCR analysis, the four loci were assigned to chromosomes 2 (MAP‐2), 9 (PID‐1), 1 (PID‐2) and 8 (PID‐3), respectively. These chromosomes carry numerous other previously determined viral integration and chromosomal fragile sites and the myc oncogenes. The PID‐1 locus was further found in Southern analysis to be rearranged and amplified in another cervical cancer biopsy and a cervical carcinoma cell line (CaSki). On Northern analysis, the PID‐1 and ‐3 probes detected a 3.0‐ and a 3.6‐kb transcript, respectively, in normal cervical cells and in cervical cancer cell lines. The findings suggest that HPV‐16 genome integrates frequently into topologically destabilized and transcriptionally active chromosomal sites. It remains to be elucidated whether the MAP‐2 and the PID loci contribute to the pathogenesis of cervical cancer.

Detection of mutations in the p53 gene in human head and neck carinomas by single strand conformation polymorphism analysis

Using the polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP) analysis, we have examined the highly conserved regions of the p53 gene in 58 biopsy samples of head and neck tumors. Mutations were found in 13/58 (23%) tumor specimens, but not in 6 normal tissues. Ten of 13 mutations were due to single base changes and the remaining 3 were 1- or 8-base deletion mutants. These mutations were clustered in exons 5 and 7 and resulted in amino acid changes. Our results seem to indicate that mutations in the p53 gene contribute to a significant number of cases of the head and neck tumors including 20% of nasopharyngeal carcinoma biopsies. The relationship of Epstein-Barr virus or human papillomavirus and p53 gene mutations in this group of cancers was also analyzed and discussed.

Jun-B oncogene aberrations in cervical cancer cell lines

We describe here structural and expression analysis of the jun-B oncogene in two cervical cancer cell lines. In the CC7T-a cell line, results from both Southern analysis and cDNA cloning studies revealed the existence of two structurally altered jun-B alleles besides the normal gene. One of the altered alleles was due to a type 16 human papillomavirus (HPV-16) integration event, whereas the other allele was a consequence of a chromosomal translocation involving chromosome 19 (jun-B) and an EST182 locus residing in chromosome 15. In the HeLa cell line, which contains integrated HPV-18, an apparent structural aberration, a 3-fold amplification and a 3-fold overexpression of the jun-B gene were observed. Our observations suggest that deregulation of the jun-B gene expression may have contributed to the transformation process in these two cervical cancer cell lines.

Frequent deletions and sequence aberrations at the transgene junctions of transgenic mice carrying the papillomavirus regulatory and the SV40 TAg gene sequences.

Exogenous DNA microinjected into one-cell mouse zygotes either integrates into the host genome within a short time span, or is rapidly degraded. On integration, a transgene squence is frequently reiterated. In this report, we describe the enzymatic amplification analysis of transgene junctions of 12 transgenic mice carrying different copy numbers of the same transgene with dissimilar ends. The transgene was composed of the regulatory sequence of the type 18 human papillomavirus linked to the TAg gene of the SV40 virus. Nucleotide sequences of 36 of these junctions were also determined. Deletions were found in 33 (91.7%) of the junctions analysed. At the crossover regions, 55.6% contained short overlapping sequences of one to six nucleotides. Insertions of 2–6 extraneous nucleotides were also found in 8.3% of the transgene junctions. Within a 10-nucleotide sequence on both sides of the transgene junctions, topoisomerase I (topo I) cleavage sites, runs of homogeneous purines or pyrimidines, alternating purine-pyrimidine tracks and (A-T)-rich sequences were found frequently. Stringent control experiments were also performed to ascertain that the observations made were not artefacts resulting from the polymerase chain reaction. Our data therefore indicate that damage had occurred quite frequently and extensively in our transgene construct. Such transgene damage may also occur to various extents in mice carrying other transgenes. Primary structure of the nucleotide sequences of the injected DNA seems to influence the process of transgene reiteration and aberration.

FZD1 activates protein kinase C delta-mediated drug-resistance in multidrug-resistant MES-SA/Dx5 cancer cells

Multidrug-resistant (MDR) cancer is a major clinical problem in chemotherapy of cancer patients. We have noted inappropriate PKCδ hypomethylation and overexpression of genes in the PKCδ/AP-1 pathway in the human uterus sarcoma drug-resistant cell line, MES-SA/Dx5 cells, which also overexpress p-glycoprotein (ABCB1). Recent studies have indicated that FZD1 is overexpressed in both multidrug-resistant cancer cell lines and in clinical tumor samples. These data have led us to hypothesize that the FZD1-mediated PKCδ signal-transduction pathway may play an important role in drug resistance in MES-SA/Dx5 cells. In this work, the PKCδ inhibitor Rottlerin was found to reduce ABCB1 expression and to inhibit the MDR drug pumping ability in the MES-SA/Dx5 cells when compared with the doxorubicin-sensitive parental cell line, MES-SA. PKCδ was up-regulated with concurrent up-regulation of the mRNA levels of the AP-1-related factors, c-JUN and c-FOS. Activation of AP-1 also correlated with up-regulation of the AP-1 downstream genes HGF and EGR1. Furthermore, AP-1 activities were reduced and the AP-1 downstream genes were down-regulated in Rottlerin-treated or PKCδ shRNA-transfected cells. MES-SA/Dx5 cells were resensitized to doxorubicin-induced toxicity by co-treatment with doxorubicin and Rottlerin or PKCδ shRNA. In addition, cell viability and drug pump-out ability were significantly reduced in the FZD1 inhibitor curcumin-treated and FZD1 shRNA-knockdown MES-SA/Dx5 cells, indicating involvement of PKCδ in FZD1-modulated ABCB1 expression pathway. Taken together, our data demonstrate that FZD1 regulates PKCδ, and the PKCδ/AP-1 signalling transduction pathway plays an important role in drug resistance in MES-SA/Dx5 cells.

Analysis of the structure and expression of the c-myc oncogene in cervical tumor and in cervical tumor-derived cell lines

The structure of the c-myc oncogene in 17 cervical tumors and patient-matched nontumor tissues from Chinese patients residing in Taiwan was analysed. In contrast to recent reports on Mexican patients, none of the samples showed rearrangements and sequence amplification in the c-myc gene. The discrepancy may be explained by different carcinogenesis mechanisms being in operation in different geographic regions. Although no structural alterations in the c-myc gene were found in seven cervical carcinoma cell lines analysed, Northern blot analysis indicated different levels of c-myc gene expression which may be related to the presence of human papillomavirus (HPV) sequence in the cell and suggests a possible c-myc-hpv interaction in some stages of the transformation process.

Mesenchymal stem cell-based HSP70 promoter-driven VEGFA induction by resveratrol alleviates elastase-induced emphysema in a mouse model.

Chronic obstructive pulmonary disease (COPD) is a sustained blockage of the airways due to lung inflammation occurring with chronic bronchitis and/or emphysema. Progression of emphysema may be slowed by vascular endothelial growth factor A (VEGFA), which reduces apoptotic tissue depletion. Previously, authors of the present report demonstrated that cis-resveratrol (c-RSV)-induced heat-shock protein 70 (HSP70) promoter-regulated VEGFA expression promoted neovascularization of genetically modified mesenchymal stem cells (HSP-VEGFA-MSC) in a mouse model of ischemic disease. Here, this same stem cell line was evaluated for its protective capacity to alleviate elastase-induced pulmonary emphysema in mice. Results of this study showed that c-RSV-treatment of HSP-VEGFA-MSC exhibited synergy between HSP70 transcription activity and induced expression of anti-oxidant-related genes when challenged by cigarette smoke extracts. Eight weeks after jugular vein injection of HSP-VEGFA-MSC into mice with elastase-induced pulmonary emphysema followed by c-RSV treatment to induce transgene expression, significant improvement was observed in respiratory functions. Expression of VEGFA, endogenous nuclear factor erythroid 2-related factor (Nrf 2), and manganese superoxide dismutase (MnSOD) was significantly increased in the lung tissues of the c-RSV-treated mice. Histopathologic examination of treated mice revealed gradual but significant abatement of emphysema and restoration of airspace volume. In conclusion, the present investigation demonstrates that c-RSV-regulated VEGFA expression in HSP-VEGFA-MSC significantly improved the therapeutic effects on the treatment of COPD in the mouse, possibly avoiding side effects associated with constitutive VEGFA expression.

Analysis of the unoccupied site of an integrated human papillomavirus 16 sequence in a cervical carcinoma

We have previously cloned and analyzed the structure of a type 16 human papillomavirus (HPV16) integration in a primary cervical carcinoma tissue, M50 (Choo et al., J. Virol. 62, 1659-1666, 1988). We found that specific nucleotide sequences within the HPV16 genome influenced the genomic organization of the integrated viral genome. Using the viral-cellular junctions of the M50 DNA as probes, we have now cloned the unoccupied site from a human genomic library. Mapping analysis showed that a deletion of about 1.1 kilobase pairs (kb) had occurred at the integration site of M50. Sequencing of the integration junctions of the unoccupied site and comparison with the viral sequence has revealed short regions of sequence homology between the viral and the cellular genomes at both junctions. Our results are consistent with a mechanism of integration of the HPV16 sequences in the M50 carcinoma involving illegitimate recombination events using short patches of homologous sequences between the two heterologous genomes for anchorage and as guides for crossover. Preferred topoisomerase I cleavage sites and alternating purine and pyrimidine bases, which favor the formation of Z-DNA, could also be identified at the integration regions, supporting a proposed role for the topoisomerase I enzyme in the illegitimate recombination in the viral integration process.