Impact of Grade, Hormone Receptor, and HER-2 Status in Women with Breast Cancer on Response to Specific Chemotherapeutic Agents by in vitro Adenosine Triphosphate-based Chemotherapy Response Assay

This study was designed to assess whether histological and biological factors of breast cancer can predict chemoresponse to specific agents. Adenosine triphosphate-based chemotherapy response assay (ATP-CRA) was employed to retrieve chemoresponse to 5-fluorouracil (5-FU), doxetaxel, doxorubicin, epirubicin, and paclitaxel in 49 patients. Tumors with high histologic and nuclear grade have higher response rate to doxorubicin (P<0.05) and palitaxel (P<0.05). Estrogen receptor (ER)-negative tumors respond well to doxorubicin (P=0.038), and progesterone receptor (PR)-negative tumors to 5-FU (P=0.039), doxetaxel (P=0.038), doxorubicin (P=0.000), epirubicin (P=0.010), and paclitaxel (P=0.003). Among the breast cancer subtypes determined by ER, PR, and HER-2 immunohistochemical stains, the HER-2+/ER- subtype has a higher response rate to doxorubicin (P=0.008). This in vitro result suggests that the combination of histologic and nuclear grade, hormone receptor, and HER-2 status can be a predictive factor of response to specific chemotherapy agents. Further in vivo study should be followed for clinical trials.

Effect of Surgical Therapy on the Courses of Human Papillomavirus Infection in Cervical Intraepithelial Neoplasia / 대한산부인과학회잡지

OBJECTIVE: To investigate whether human papillomavirus (HPV) associated with cervical intraepithelial neoplasia was successfully eliminated after surgical therapy such as large loop excision of transformation zone (LLETZ) or type I extended hysterectomy. METHODS: Seventy four cervical intraepithelial neoplasia (CIN) patients with HPV infection who were admitted for LLETZ or type I extended hysterectomy were recruited into this prospective study. HPV infection was confirmed by HPV DNA genotyping using HPV DNA Chip (Biomedlab, Seoul, Korea) before and after surgical therapy. RESULTS: According to the assay used (HPV DNA Chip(R)) at the postoperative visit, 82.5% of treated patients were completely free from HPV infection. And there was no statistical difference between LLETZ and type I extended hysterectomy (p=0.452). However, there was persistent infection in 17.5% of treated patients after final surgical therapy. CONCLUSION: Although the HPV infection associated with CIN was effectively eliminated after LLETZ and type I extended hysterectomy, there was persistent infection in 17.5% of treated patients. Therefore, the careful followed-up examination of HPV infection should be taken in these patients.

Effect of Ganciclovir on Growth of Neuroblastoma Cell Line Expressed HSV-TK Gene / 대한소아혈액종양학회지

BACKGROUND: Replication of defective adenoviral vectors can be used for gene transfer into a wide spectrum of replicating and nonreplicating cells. Accordingly, selective introduction of genes encoding for susceptibility to nontoxic drugs into proliferating tumor may be used to treat cancer. We investigated the efficacy of in vitro transduction of neuroblastoma cell with the herpes simplex virus thymidine kinase gene followed by administration of the antiviral drug ganciclovir. METHODS: The recombinant adenoviral vector ADV/TK carrying the HSV-TK gene, under the control of the promoter from Rous sarcoma virus long term terminal repeat was constructed. And 1 x 10(4) Neuro 2a cells were plated in 96 well cultured plates and infected with ADV/TK at multiplicity of infection of 0, 1, 10, and 100. Twenty-four hours later, the infected cells were treated with PBS or ganciclovir at a concentration of 10 g/ml. After 48hr, the surviving cells in 96 well plates were determined by MTT assay. RESULTS: After infection in vitro with ADV/TK at moi of 0, 1, 10, 100 and subsequent ganciclovir treatment, the percent survival rate of 1 x 10(4) Neuro 2a cells were 105%, 32%, 25%, and 15%. But the survival rate of 1 x 10(4) Neuro 2a cells with PBS treatment were 100%, 92%, 105%, 103%. CONCLUSION: We concluded that in vitro transduction of neuroblastoma cell with the herpes simplex-thymidine kinase gene followed by administration of the antiviral drug ganciclovir is very effective.

Generation of Monoclonal Antibodies Against Human Papillomavirus Type16 E7 Protein : Usefulness for Various E7 Detection Systems

The gene encoding E7 oncoprotein of human papillomavirus type 16 was cloned and expressed in Escherichia coli, and the monoclonal antibodies (Mabs) against this expressed protein were generated. For the efficient immunization, two kind of recombinant E7 protein in fusion form were produced. One was maltose binding protein (MBP) fusion type (MBP-E7) and the other was T7 phage gene 10 product fusioa type (gene 10-E7). Immunization with these two fusion protein to mice, finally two Mabs (VD6 and IB10) were obtained. VD6 and IB10 showed reactivities with E7 protein in CaSki cell but not in HeLa by Western blot analysis. In addition, the Mab, VD6, reacted with COS-7 cell transfected with E7 gene majorly in cytoplasm by immunofluorescence test. Also VD6 could detect E7 protein in cytoplasm and nucleus of CaSki ceU by immunogold electron microscopy. Based on these results, the Mab VD6 was could be used for various E7 detection system such as Western blot analysis and immunohistochemical methods.

The effects of wild type p53 tumor suppressor gene expression on the normal human cervical epithelial cells or human epidermal keratinocytes transformed with human papillomavirus type 16 DNA

The inactivation of p53 and p105RB by viral proteins or by mutations plays a key role in the oncogenesis of cervical carcinoma. The E6 and E7 proteins of HPV type 16 can bind to p53 and p105RB tumor suppressor gene products, respectively. In the present study, we tested a simple in vivo model that could explain the interactions between HPV E6 oncoprotein and p53 tumor suppressor protein. Our results showed that the life span of normal cervical epithelial cells was increased up to 4.5 times when transfected with expression vector containing E6/E7 ORF of HPV type 16. However, these cells did not divide after second crisis. Therefore, we employed an established human epidermal keratinocytes, RHEK-1. When transfected with an expression vector containing E6 ORF of HPV type 16, RHEK-1 cells showed anchorage independent growth character. When RHEK-E6 cells were transfected with wild type p53 expression vector, the growth rate of the RHEK-E6 cells was diminished. After 48 hours of transfection, many cells showed apoptotic signal but no more apoptotic signal was observed thereafter. These results suggested that the overexpression of the wild type p53 could overcome the dysfunction of the p53 on the cell cycle regulation imposed by E6 protein although not being of physiological condition.

Analysis of p53 tumor suppressor gene mutations and human papillomavirus infection in human bladder cancers

To determine whether the dysfunction of p53 caused either by mutation of the p53 gene itself or by binding to E6 protein of oncogenic HPVs is involved in the transitional cell carcinomas (TCCs) of the bladder, we analyzed 23 TCCs of the bladder. DNA was extracted from each paraffin embedded tissue of TCCs of bladder and polymerase chain reaction (PCR)/single strand conformation polymorphism (SSCP) analysis were performed to screen mutations in p53 tumor suppressor gene, then PCR/dot blot hybridization were performed to detect infection of HPVs. We found that p53 gene mutation was found in 3 cases and oncogenic HPV infection was detected in 8 cases and thus, the overall incidence of possible p53 dysfunction was 47.8% on DNA analysis (If the results of immunohistochemistry to detect overexpression of p53 protein were included, the incidence was 60.9%). Therefore, we concluded that dysfunction of p53 plays a major role in the development of TCCs of bladder in Korean patients.

Role of human papillomavirus and p53 tumor suppressor gene in cervical carcinogenesis

To determine whether the dysfunction of p53 protein, caused either by the mutation of p53 gene itself or by the human papillomavirus (HPVs) is involved in the development of cervical cancers and to find out the status of p53 tumor suppressor gene in HPV positive or negative cancers, we analyzed 64 cases of primary cervix cancers. First, polymerase chain reaction (PCR) was performed with E6 consensus primer pairs to detect the infection of HPVs in cervix cancer tissues. Second, to screen the p53 gene mutation, PCR of p53 exon 5 through 9 and single strand conformation polymorphism (SSCP) analysis were performed with p53 exon specific primers. Finally, overexpression of p53 protein was checked by immunohistochemical staining with anti p53 antibody. We found HPVs in 43 cases out of 64 cases (67.2%). HPV type 16 was positive in 26 cases, type 18 was positive in 7 cases, and 6 cases were positive for both HPV type 16 and 18. HPV type 31 or 33 was not detected in our experiments, and in 4 cases, type of HPVs were unknown. The SSCP analysis showed mobility shifts in 8 cases (6 in HPV positive cases; 2 in HPV negative cases) and the sequence analysis confirmed the results of SSCP analysis. In addition, the overexpression suggesting the mutation of p53 gene was detected in 27 cases (42.2%, 21 in HPV positive cases; 6 cases in HPV negative cases). Our results support the previous reports that the dysfunction of p53 plays an important role in the development of cervix cancers but contrary to the results obtained from cervix cancer all lines, there is no inverse correlation between HPV infections and p53 mutations in primary cervix cancers.

Expression and localization of human papillomavirus type 16 E6 and E7 open reading frame proteins in human epidermal keratinocyte

Over 60 different types of human papillomavirus (HPV) have been identified, and they are classified into high and low risk groups based on the risk for malignant progression of HPV associated lesions. HPVs belonging to a high risk group have been shown to express two major transforming proteins, E6 and E7. With respect to the transforming activity of these proteins, many investigators have reported the location of these proteins in the cell, but their results are still controversial. In the present study, HPV type 16 E6 or E7 open reading frame (ORF) proteins were expressed and localized in human epidermal keratinocytes (RHEK-1) using the vaccinia virus as an expression vector. Immunofluorescence detection using monoclonal antibodies against E6 or E7 ORF proteins revealed that E6 or E7 proteins of HPV type 16 were located in the cytoplasm of RHEK-1 cells. These results suggest that E6 and E7 proteins bind to the tumor suppressor counterparts, thereby preventing transport of these proteins into the nucleus. These antioncogene products that fail to be rapidly transported out of the cytosol may be degraded by certain proteases such as the ubiquitin dependent system. In this way, the precise function of antioncogene products in the regulation of cell growth could be destroyed, and abnormal cell growth could occur.

Mutations in the pre-core region of hepatitis B virus DNA in patients with chronic liver diseases

To investigate the prevalence of point mutation in the pre-core (pre-C) region of hepatitis B virus (HBV) DNA, we performed dot blot hybridization and sequencing of enzymatically amplified HBV DNA from the sera of 25 patients with HBeAg-positive and 32 patients with HBeAg-negative chronic liver diseases. The pre-C region of HBV DNA was successfully amplified by polymerase chain reaction (PCR) from 55 (96.5%) of 57 sera. According to the status of serum HBeAg, HBV DNA was amplified from all 25 sera of HBeAg-positive patients and 30 (93.8%) of 32 sera of HBeAg-negative patients. All amplified DNA from the sera of 25 patients with HBeAg-positive and that from 28 (93.3%) of 30 patients with HBeAg-negative chronic liver diseases hybridized with the wild type probe. In addition, that from 5 (20.0%) among 25 patients with HBeAg-positive and 16 (53.3%) among 30 patients with HBeAg-negative chronic liver diseases hybridized also with the mutant type probe. These results suggest that the prevalence of point mutation in the pre-C region of HBV DNA is relatively high in patients with HBeAg-negative chronic liver diseases and further study is mandatory to identify the significance of this mutation.

Identification of mutagenic site of c-H-ras oncogene damaged by N-acetoxyacetylaminofluorene(AAAF)

A molecularly cloned human cellular H-ras (c-H-ras) oncogene(pbc N1 plasmid) was treated with N-acetoxyacetylaminofluorene (AAAF) in vitro and subcloned into E.coli. This was done to identify the mutational changes at specific codons of the gene. Guanine nucleotides were identified as the major AAAF binding site of the DNA adduct formed. Base changes in codons 12 and 61 were determined by the analysis of restriction fragment length polymorphism (RFLP) and site specific oligonucleotide hybridization. RFLP was observed due to the loss of the Hpall recognition site at codon 11 and 12 of AAAF-treated c-H-ras gene. Hybridization of AAAF treated c-H-ras with 32P-labeled oligonucleotide probes for the mutant alleles of codon 61 showed no substitutions at codon 61. From these results, it is assumed that AAAF treatment in vitro caused mutation at codon 12 but not at codon 61 of the c-H-ras oncogene and that codon 12 is the primary target of mutation by AAAF