白澤 浩

[原著] Sakao S, Hashimoto T, Shino Y, Saito T, Kado S, Sashiyama H, Tatsumi K, Kuriyama T, Shirasawa H.: Expression of the potential novel gene E6DG1 downregulated by the E6 protein of human papillomavirus type 16 is correlated with anchorage-independent growth

A novel gene transcript that is downregulated by HPV16 E6 protein was identified in mouse cells using differential hybridization and designated E6DG1. The cloned cDNA of E6DG1 was 1.3 kb in length and contained a small ORF potentially encoding a polypeptide of 45 amino acids. In vitro transcription and translation of E6DG1 cDNA resulted in a product of approximately 7 kDa and Western blot analysis using antibodies for E6DG1 peptide detected 7 and 14 kDa proteins. Downregulation of E6DG1 mRNA levels in cells expressing HPV16 E6 protein was observed at subconfluent cell densities, but not in confluent cells. Repression of E6DG1 protein enhanced the anchorage- independent growth and weakened the cell adhesion in Panc 1 cells. Immunofluorescence analysis revealed the localization of E6DG1 within the nucleus. These results indicate that the E6DG1 protein may function in a signaling pathway related to anchorage-independent growth and adhesion control.

Human pancreatic cancers harbor mutations in the K-ras gene, and these mutations convert the gene oncogenic and constitutively active forms. However, in pancreatic cancer cells little is known about the activation of the downstream pathways of Ras, MEK - ERK and MEKK1 - JNK, and their roles in cell survival and proliferation. An analysis of nine pancreatic cancer tissues revealed JNK activation in all tumor samples and ERK activation in three tumor samples. Colony formation assays by transfection of dominant negative mutants of Ras, ERK or MEKK1 into pancreatic cancer cell lines (BxPC-3, PANC-1, MIAPa-Ca-2 and AsPC-1) and an amnion-derived cell line (FL) revealed that DN - MEKK strongly inhibits the survival of colonies in pancreatic cancer cells, but not in FL cells. In vitro kinase assays and luciferase assays using the Gal4c-Jun system revealed that in pancreatic cancer cells DN-MEKK fails to inhibit JNK activation. In PANC-1 cells, c-Jun was found to be a major component of protein component binding to AP-1 site and CRE, but not in FL cells. The inhibitory effect of DN-MEKK in PANC-1 cells was thought to be the result of the inhibition of c-Jun DNA-binding. The difference of suppression in pancreatic cancer cells and non-pancreatic cancer cells suggested that the MEKK1 pathway mainly contributes to cell survival in pancreatic cancer cells and may provide an advantage for the gene therapy of pancreatic cancers using DN - MEKK expression vectors.

Human pancreatic cancers harbor mutations in the K-ras gene, and these mutations convert the gene oncogenic and constitutively active forms. However, in pancreatic cancer cells little is known about the activation of the downstream pathways of Ras, MEK - ERK and MEKK1 - JNK, and their roles in cell survival and proliferation. An analysis of nine pancreatic cancer tissues revealed JNK activation in all tumor samples and ERK activation in three tumor samples. Colony formation assays by transfection of dominant negative mutants of Ras, ERK or MEKK1 into pancreatic cancer cell lines (BxPC-3, PANC-1, MIAPa-Ca-2 and AsPC-1) and an amnion-derived cell line (FL) revealed that DN - MEKK strongly inhibits the survival of colonies in pancreatic cancer cells, but not in FL cells. In vitro kinase assays and luciferase assays using the Gal4c-Jun system revealed that in pancreatic cancer cells DN-MEKK fails to inhibit JNK activation. In PANC-1 cells, c-Jun was found to be a major component of protein component binding to AP-1 site and CRE, but not in FL cells. The inhibitory effect of DN-MEKK in PANC-1 cells was thought to be the result of the inhibition of c-Jun DNA-binding. The difference of suppression in pancreatic cancer cells and non-pancreatic cancer cells suggested that the MEKK1 pathway mainly contributes to cell survival in pancreatic cancer cells and may provide an advantage for the gene therapy of pancreatic cancers using DN - MEKK expression vectors.

Human severe combined immune deficiency (SCID) syndrome is a heterogeneous disease, and some SCID mutations are characterized by a high sensitivity to ionizing radiation (RS-SCID). This phenomenon is suspected to be due to impaired repair systems for damaged DNA [J. Exp. Med. 188 (1998) 627]. However, the details are not fully understood. In this work, two RS-SCID cell lines were conferred radioresistance by infection with a recombinant adenovirus encoding E6/E7 (AxE67), and by transfection with an expression vector bearing human telomerase reverse transcriptase (hTERT). Our findings suggest that overexpressed E6 and E7 or elevated telomerase activity have some influence on the acquired radioresistance in human RS-SCID cells. (C) 2002 Elsevier Science B.V All rights reserved.

Transduction of human papillomavirus type 16 (HPV16) E6/E7 into primary culture of human esophageal keratinocytes using a recombinant adenovirus prolonged the life-span, while untreated cells senesced within 14-16 population doublings (PDLs). Up-regulation of telomerase activity and acquisition of serum-resistant growth were observed in the esophageal keratinocytes with extended lifespan between 50 and 100 PDLs, and drastically increased after 100 PDLs. A keratinocyte sample with a polymorphism of Pro/Pro at codon 72 of p53 showed resistance to HPV16 E6/E7-induced life-span-extension and immortalization, in contrast to others with p53 polymorphisms of Arg/Arg or Arg/Pro, which did not. The high efficiency of E6/E7-induction by adenovirus vector also revealed the M1 and M2 stages of keratinocyte immortalization first described in this report.

[原著] Kado S, Kawamata Y, Shino Y, Kasai T, Kubota K, Iwasaki H, Fukazawa I, Takano H, Nunoyama T, Mitsuhashi A, Sekiya S, Shirasawa H. : Detection of human papillomaviruses in cervical neoplasias using multiple sets of generic polymerase chain reaction primers.

Objective. The aim of this study was to evaluate precisely the differences in the spectra of human papillomavirus (HPV) types detected by different generic primer pairs commonly used for detection of this extraordinarily heterogeneous virus. Methods. Three sets of polymerase chain reaction (PCR) primers for the L1 open reading frame (ORF) and two sets for E6/E7 ORFs were used to detect HPVs in DNAs from 107 cervical tissues, including 77 cervical neoplasias. HPV types were determined by analysis of restriction fragment length polymorphisms (RFLPs) and nucleotide sequencing. Results. A high overall detection rate of HPV in cervical neoplasias (76/77, 98.7%) was achieved by polymerase chain reaction (PCR) amplification with multiple sets of generic primers, while the detection rate for each individual primer pair varied from 48/77 (62%) to 70/77 (91%). Only in 34 of 77 cases (44%) were HPV DNAs positive for all sets of primer pairs. Further determination of HPV types by RFLPs and nucleotide sequencing showed inconsistencies between the PCR primer pairs used. Conclusion. Our study revealed that the HPV detection rate is critically affected by the choice of PCR primers, and that appropriate use of combinations of generic PCR primer sets followed by RFLP analyses is both necessary and sufficient for typing most HPVs in cervical lesions. More precise methods such as sequencing would be necessary in only a few cases. (C) 2001 Academic Press.

A substantial fraction of neuroblastomas found by mass screening have been suggested to regress spontaneously because of the high incidence of infantile neuroblastomas in the screening population. In this study, 70 neuroblastomas were analyzed for expression of proto-oncogenes related to neuronal differentiation to clarify the biological significance of proto-oncogene expression in the screening-positive and -negative tumors. The tumors consisted of 39 neuroblastomas found by screening (group 1), 16 non-N-myc-amplified nenroblastomas found by clinical symptom(s) (group 2), and 15 N-myc-amplified neuroblastomas found by clinical symptom(s) (group 3), The expression of c-spc, trk A, and N-myc in tumor tissues was analyzed by quantitative RNA PCR, Neuronal c-srcN2 expression varied significantly in the following order: group 1 > group 2 > group 3, The level of expression of trk A was markedly reduced in group 3 but did not differ in groups 1 and 2, Most tumors in group 3 overexpressed N-myc, However, N-myc expression in group 1 was significantly higher than that in group 2, Thus, the characteristics of proto-oncogene expression in screening-positive tumors included enhanced expression of c-srcN2 and N-myc mRNA, regardless of nonamplification of N-myc, Our results suggest that the role of N-myc differs in neuroblastomas detected by screening and in N-myc-amplified tumors.

A substantial fraction of neuroblastomas found by mass screening have been suggested to regress spontaneously because of the high incidence of infantile neuroblastomas in the screening population. In this study, 70 neuroblastomas were analyzed for expression of proto-oncogenes related to neuronal differentiation to clarify the biological significance of proto-oncogene expression in the screening-positive and -negative tumors. The tumors consisted of 39 neuroblastomas found by screening (group 1), 16 non-N-myc-amplified nenroblastomas found by clinical symptom(s) (group 2), and 15 N-myc-amplified neuroblastomas found by clinical symptom(s) (group 3), The expression of c-spc, trk A, and N-myc in tumor tissues was analyzed by quantitative RNA PCR, Neuronal c-srcN2 expression varied significantly in the following order: group 1 > group 2 > group 3, The level of expression of trk A was markedly reduced in group 3 but did not differ in groups 1 and 2, Most tumors in group 3 overexpressed N-myc, However, N-myc expression in group 1 was significantly higher than that in group 2, Thus, the characteristics of proto-oncogene expression in screening-positive tumors included enhanced expression of c-srcN2 and N-myc mRNA, regardless of nonamplification of N-myc, Our results suggest that the role of N-myc differs in neuroblastomas detected by screening and in N-myc-amplified tumors.

Objective. A case of well-differentiated villoglandular adenocarcinoma of the uterine cervix, which was positive for human papillomavirus type 18, was reported. Methods. The patient was a 52-year-old multipara who was referred to our department because of an abnormal Papanicolaou smear. A 4.0-cm exophytic lesion involving the cervix was detected. She was staged as FIGO IIa and radical hysterectomy combined with bilateral pelvic lymphadenectomy was performed. In addition to histopathological examination of the resected tumor, immunohistochemical studies of estrogen and progesterone receptors were performed using monoclonal antibodies. Detection of human papillomavirus DNA was attempted by polymerase chain reaction using consensus primers. Results. The tumor was a typical well-differentiated villoglandular adenocarcinoma involving the vaginal wall. Both estrogen and progesterone receptors were negative, Human papillomavirus type 18 DNA was detected in the resected tumor. Conclusion. This is the first report of a case of typical well-differentiated villoglandular adenocarcinoma which was positive for human papillomavirus. (C) 2000 Academic Press.

Objective. A case of well-differentiated villoglandular adenocarcinoma of the uterine cervix, which was positive for human papillomavirus type 18, was reported. Methods. The patient was a 52-year-old multipara who was referred to our department because of an abnormal Papanicolaou smear. A 4.0-cm exophytic lesion involving the cervix was detected. She was staged as FIGO IIa and radical hysterectomy combined with bilateral pelvic lymphadenectomy was performed. In addition to histopathological examination of the resected tumor, immunohistochemical studies of estrogen and progesterone receptors were performed using monoclonal antibodies. Detection of human papillomavirus DNA was attempted by polymerase chain reaction using consensus primers. Results. The tumor was a typical well-differentiated villoglandular adenocarcinoma involving the vaginal wall. Both estrogen and progesterone receptors were negative, Human papillomavirus type 18 DNA was detected in the resected tumor. Conclusion. This is the first report of a case of typical well-differentiated villoglandular adenocarcinoma which was positive for human papillomavirus. (C) 2000 Academic Press.

[原著] Kado S, Kawamata Y, Shino Y, Kasai T, Kubota K, Iwasaki H, Fukazawa I, Takano H, Nunoyama T, Mitsuhashi A, Sekiya S, Shirasawa H.: Detection of human papillomaviruses in cervical neoplasias using multiple sets of generic polymerase chain reaction primers.

The prognoses of pancreatic cancer patients have been miserable even after radical surgery, and adjuvant therapy is necessary to improve the surgical results, p16(INK4a) (p16) is tight-binding and inhibitory protein for cyclin-dependent kinase 4 to induce G(1) arrest of the cell cycle. p16 gene deletion is frequently identified in human pancreas cancer. The impaired gene function of p16 might be a major factor of the uncontrolled proliferation and malignancy of pancreas cancer cells. In this study, we investigated the effect of adenovirus p16 expression vector for pancreas cancer cell proliferation to clarify whether the vector might be a promising mode to assist the surgical therapy for pancreas cancer. We constructed the adenovirus p16 expression vector AdexCACSp16 by inserting p16 cDNA to a cassette cosmid containing a nearly full-length adenovirus type 5 genome with El and E3 deletions. Thereafter, we assessed the activity of AdexCACSp16 to induce p16 gene mRNA expression in pancreas cancer cell line MIAPaCa-2 and to control cell proliferation. AdexCACSp16 induced a high level of p16 gene mRNA expression in MIAPaCa-2 cells with 1 h contact to the cells. The cell proliferation was significantly suppressed by AdexCACSp16 compared with the control adenovirus group. These data indicate that AdexCACSp16 has the potential to induce p16 gene expression and control pancreas cancer cell proliferation and that the adenovirus p16 expression vector AdexCACSp16 might be a possible method of gene therapy to improve the surgical therapeutic results for pancreas cancer.

Intranuclear inclusion bodies are sometimes observed in pulmonary adenocarcinoma by light microscopy. Electron microscopic characteristics of lung cancer cells with intranuclear inclusion bodies were studied. In addition, polymerase chain reaction (PCR) was performed using primers coding for human papillomavirus (HPV) types 16, 18, and 33. Eosinophilic intranuclear inclusion bodies were observed in 22 out of 285 cases by light microscopy. Immunohistochemically, cancer cell nuclei stained with PE-10. Three types of intranuclear inclusion bodies were classified electron microscopically. Type A showed aggregation of electron dense particles (30-40 nm) with an electron-dense core and was most frequently observed. Type B consisted of a mass of branching and whirling tubular structures. Type B intranuclear inclusions had a relationship with inner nuclear membrane. In type C, several spherical inclusions were observed in one nucleus. HPV DNA was detected using PCR and type-specific probes in a case with type A inclusion bodies. This study suggests that intranuclear inclusion bodies in pulmonary adenocarcinoma are formed by several different mechanisms.

Recently a relationship between serotonin transporter transcriptional control region (5-HTTLPR) polymorphism and anxiety related personality traits in Caucasians was reported. We performed PCR of DNAs from the blood for determining the 5-HTTLPR genotypes of 191 Japanese subjects, which were medical staff and students, and obtained Revised NEO Personality Inventory (NEO-PI-R) and the Temperament and Character Inventory (TCI) in 144 subjects. The association was analyzed by one-way ANOVA. The present study demonstrated that allelic frequency of 5-HTTLPR (s allele frequency was 0.785) in our subjects was considerably different from that in Caucasians. No significant differences were found in the anxiety-related personality traits among genotypes, while cooperativeness in TCI showed a significant difference among genotypes. The property of 5-HTTLPR may not be reflected directory on the personality inventories. (C) 1999 Elsevier Science ireland Ltd. All rights reserved.

Intranuclear inclusion bodies are sometimes observed in pulmonary adenocarcinoma by light microscopy. Electron microscopic characteristics of lung cancer cells with intranuclear inclusion bodies were studied. In addition, polymerase chain reaction (PCR) was performed using primers coding for human papillomavirus (HPV) types 16, 18, and 33. Eosinophilic intranuclear inclusion bodies were observed in 22 out of 285 cases by light microscopy. Immunohistochemically, cancer cell nuclei stained with PE-10. Three types of intranuclear inclusion bodies were classified electron microscopically. Type A showed aggregation of electron dense particles (30-40 nm) with an electron-dense core and was most frequently observed. Type B consisted of a mass of branching and whirling tubular structures. Type B intranuclear inclusions had a relationship with inner nuclear membrane. In type C, several spherical inclusions were observed in one nucleus. HPV DNA was detected using PCR and type-specific probes in a case with type A inclusion bodies. This study suggests that intranuclear inclusion bodies in pulmonary adenocarcinoma are formed by several different mechanisms.

Advanced neuroblastoma and malignant liver tumor are representative childhood cancers for which combined chemotherapy including cisplatin and doxorubicin is routinely performed, The prognosis of patients with tumors which develop multiple drug resistance (MDR) is unfavorable. To elucidate the role of multidrug resistance-associated protein (MRP) and canalicular multispecific organic anion transporter (cMOAT) in the clinical behavior of the tumors, we examined 42 neuroblastomas and 10 malignant liver tumors for the expressions of MRP and cMOAT by quantitative RNA-polymerase chain reaction (PCR). The amplification and expression of N-myc oncogene in the neuroblastomas were also investigated, We found a close association between MRP and N-myc expression in each neuroblastoma sample but no significant relationship between MRP expression and the patients' outcome, The forced expression of N-myc failed to enhance the expression of MRP in N-myc transfected neuroblastoma cell lines, cMOAT was rarely expressed in the neuroblastomas, but was frequently expressed in the malignant liver tumors. The expression of MRP and cMOAT in the childhood liver tumors was more common and higher, especially in advanced cases with a poor outcome, than that observed in normal liver or in 9 hepatocellular carcinomas from adult patients. The enhanced expression of these genes might be characteristic of childhood malignant liver tumors and related to their clinical chemoresistance.

A new cell line, Yumoto, derived from a squamous cell carcinoma of the uterine cervix, was established from serially transplanted tumor tissues in nude mice. Monolayer cultured cells were polygonal and formed pavement-like sheet. They showed a piling-up tendency and were devoid of contact inhibition. Electron micrographs demonstrated the presence of microvilli on the cell surface, abundant tonofilaments in the cytoplasm, and the connection with desmosomes. These electron micrographical characteristics of Yumoto cells were consistent with those of squamous cell origin. Yumoto cells were highly tumorigenic in BALB/c nude mice and produced a well-differentiated squamous cell carcinoma of keratinizing type which closely resembled to the original tumor tissues in nude mice. The presence of HPV DNA was examined using polymerase chain reaction and Southern blot analysis, but no known types of HPV DNA could be detected. Exons 2 through 11 of the p53 gene were analyzed by direct DNA sequencing, revealing a homozygous mutation at codon 281 in exon 8, GAC to CAC (Asp-->His). Furthermore, physical p53-gene deletion was demonstrated by dual-color fluorescence in situ hybridization. This cell line is useful for studying the carcinogenesis of cervical carcinoma and for investigating the biological characteristics of a HPV-negative and mutated p53 squamous cell carcinoma of the uterine cervix. (C) 1998 Academic Press.

Neuroblastomas present a wide variety of clinical and biological behaviors, which are reflected by the heterogeneous expressions of protooncogenes related to the neuronal differentiation and amplification of the N-myc gene. High expression of trk A and Ha-ras in neuroblastomas has been shown to be associated with an excellent patient outcome. We have previously reported that neuron-specific src mRNA was increased in chemically differentiated neuroblastoma cell lines and in clinically observed neuroblastomas without N-myc amplification, In the present study, to clarify both the value of neuronal c-srcN2 expression as a prognostic indicator and the significance of the coexpression of these photooncogenes, we examined the expression of 3 alternatively spliced src, trk A and Ha-res in neuroblastoma tissues from 60 patients by competitive RNA-polymerase chain reaction (PCR). The restates indicate that protooncogene expression in neuroblastomas correlated with a favorable outcome for c-srcN2 and trk A. N-myc gene was amplified exclusively in tumors with low levels of trk A. Low expression of c-srcN2 and trk A might thus characterize different aggressive phenotypes due to different signal transduction pathways of neural differentiation in neuroblastoma. The combined analyses for c-srcN2 and trk A expression by RNA-BCR should provide information about the biological phenotype of a neuroblastoma within a short period of time after obtaining tumor material. (C) 1998 Wiley-Liss, Inc.