Kei Fujinaga

The Ets-1 and Ets-2 transcription factors activate the promoters for invasion-associated urokinase and collagenase genes in response to epidermal growth factor

Urokinase plasminogen activator (uPA) has been associated with invasion and metastasis in breast cancer. The expression of uPA and 92 kDa type IV collagenase (gelatinase B/MMP‐9) is regulated by growth factors, receptor‐type tyrosine kinases and cytoplasmic oncoproteins. Here, we have identified transcriptional requirements for the induction of uPA and 92 kDa type IV collagenase by epidermal growth factor (EGF). EGF stimulates the motile and invasive activities specifically in the ErbB‐2‐overexpressing SK‐BR‐3 cells. Expression of extracellular matrix‐degrading proteases including type I collagenase/MMP‐1, 92 kDa type IV collagenase/MMP‐9, uPA and uPA receptor were induced. EGF also transiently stimulated expression of the transcription factors Ets‐1 and Ets‐2. Reporter transfection assays revealed the activation of uPA and MMP‐9 collagenase promoters by EGF and the requirement of each of the composite Ets and AP‐1 transcription factor binding sites for an EGF response. Most notably, transfections with the Ets‐1 and Ets‐2 expression vectors potentiated uPA and MMP‐9 promoter activation in response to EGF. Mutation of the threonine 75 residue of chicken Ets‐2 conserved in the Pointed group of the Ets family proteins abrogated the ability of Ets‐2 to collaborate with EGF. Ets‐1 and Ets‐2 were highly expressed in invasive breast tumor cell lines. Our results suggest that Ets‐1 and Ets‐2 provide the link connecting EGF stimuli with activation of uPA and 92 kDa type IV collagenase promoters and may contribute to invasion phenotypes. Int. J. Cancer 77:128–137, 1998.© 1998 Wiley‐Liss, Inc.

Simultaneous detection and typing of genital human papillomavirus DNA using the polymerase chain reaction

A simple method has been developed for detecting a broad range of genital human papillomavirus (HPV) types using the polymerase chain reaction (PCR). We utilized two consensus sequence primer pairs within the E6 and E7 open reading frames to amplify HPV DNA; malignant HPV DNA (from HPV-16, -18, -31, -33, -52b and -58) was amplified using the pU-1M/pU-2R primer pair whereas benign HPV DNA (from HPV-6 and -11) was amplified using the pU-31B/pU-2R primer pair. Identification of the amplification product was confirmed by restriction enzyme digestion. In this study, a pU-1M/pU-2R-mediated PCR was successfully applied to 39 cervical carcinoma specimens; HPV-16 was detected in 19 cases, HPV-18 in five cases, HPV-31 in two cases, HPV-33 in two cases, HPV-52b in one case, HPV-58 in three cases, and an unknown type(s) was detected in four cases. Overall, the prevalence of HPV was 84.6%. The results indicate that this detection system is useful for the detection of HPVs not only of known types but also of new types.

Fusion of an ETS-family gene, EIAF, to EWS by t(17;22)(q12;q12) chromosome translocation in an undifferentiated sarcoma of infancy

EIAF is a newly isolated ETS‐family gene that is located on 17q21 and codes for the adenovirus EIA enhancer‐binding protein. In our chromosome analysis of 18 of the Ewing family of tumors and undifferentiated sarcomas, we found t(17;22)(q12;q12) in an MIC2 antigen‐positive undifferentiated sarcoma of infancy. On Southern blot analysis, EWS and EIAF cDNA probes hybridized to the same rearranged band, indicating that an EWS‐EIAF fusion gene was formed in the tumor. Further Southern blot analysis using four EIAF cDNA probes of different sizes showed that the breakpoint lies in the region upstream to the ETS domain of the EIAF gene. EIAF may be the fourth ETS‐family gene to be identified forming a fusion gene with EWS. We assume that the RNA binding domain of EWS may have been replaced by the DNA binding domain of EIAF in the EWS‐EIAF fusion protein as in other fusion proteins previously characterized in Ewing sarcoma and other types of sarcomas. Genes Chromosom Cancer 15:115–121 (1996).

Detection of Human Papillomavirus DNA Sequences in Oral Squamous Cell Carcinomas and Their Relation to p53 and Proliferating Cell Nuclear Antigen Expression

Background. The etiology of oral squamous cell carcinoma (SCC) is still obscure. Since human papillomavirus (HPV) DNAs are associated with carcinoma of the uterine cervix, carcinomas of the oral cavity were investigated to ascertain if these viruses are present in squamous carcinomas of this anatomic site.

Structure and gene organization in the transforming Hind III-G fragment of Ad12

The nucleotide sequence of the transforming Hind III-G fragment of Ad12 DNA which encompasses the left 6.8% of the genome has been determined. The fragment was 2320 nucleotides long, and contained a GC cluster at positions 126-155 and a region extremely rich in AT at positions 1098-1142 (number from the leftmost end). Possible coding regions for the two transforming gene products were assigned. The predicted coding region for T antigen g is positions 502-1069 and positions 1144-1373, which are joined by splicing (266 amino acid residues, 30 kd), and that for T antigen f is positions 1845-2126 (94 amino acid residues, 10 kd). The sequence of the Hind III-G fragment was compared with that of the transforming DNA fragment of Ad5 which encompasses the left 8.0% of the genome (2809 nucleotides). There are several discrete regions with significant sequence homology. The comparison suggests that the regions in the left two thirds of the Ad5 and Ad12 transforming DNA fragments (map units 0-4.7% in Ad5 and 0-4.4% in Ad12) bear some resemblance in their gene organizations, and code for proteins containing structurally homologous regions.

Establishment and characterization of rat cell lines transformed by restriction endonuclease fragments of adenovirus 12 DNA

Abstract Rat cells (3Y1) were transformed by the Hin dIII-G fragment of adenovirus 12 DNA and cloned in solft agar cultures, and transformed cell lines (GY1, GY2, and GY3 cells) were established. Part of the Hin dIII-G fragment of adenovirus 12 DNA was the only viral DNA sequence present in GY1 cells. The characteristics of CY1 (a rat cell line transformed by the Eco RI-C fragment of adenovirus 12 DNA), GY1, and WY3 cells (a rat cell line transformed by the whole viral DNA of adenovirus 12) were examined. The growth of each transformed cell line was unlimited as compared to that of untransformed 3Y1 cells. Complement fixation and immunofluorescence suggested that CY1 and GY1 cells contained T antigen, while WY3 cells contained both T antigen and DNA-binding protein. CY1, GY1, and WY3 cells induced tumors in rats after transplantation. In the serum of rats bearing CY1 or GY1 cell tumors only antibody to T antigen was detectable, while antibodies to both T antigen and virus-induced DNA-binding protein were present in the serum of rats bearing WY3 cell tumors. These results show that the transforming gene is located on the left end (7.2%) of adenovirus 12 DNA and suggest that T antigen is among the gene products coded for by this region of the genome.

OUTBREAK OF INFANTILE GASTROENTERITIS DUE TO TYPE 40 ADENOVIRUS

Genetic and antigenic characterisation was performed on a strain of adenovirus (EAd) isolated from an outbreak of gastroenteritis which occurred in an orphanage in the City of Sapporo, in the room housing the eldest children, who ranged in age from 14 to 22 months. 7 of the 11 children housed in that room had diarrhoea between July 11 and July 22, 1982. All 7 shed adenoviruses detectable by electron microscopy in their stools. Immune electron microscopy showed that all patients as well as the healthy contacts sharing the room underwent seroconversion to EAd. There was no homology, or very slight homology, between DNA of EAd and those of adenoviruses belonging to subgroups A to E. Antigenically EAd was closely related to type 40 adenovirus, so far the sole member of the newly identified subgroup F. This outbreak of gastroenteritis is the first in which the causative agent has been identified as being a member of subgroup F adenoviruses.

Human papillomavirus type 18 DNA and E6-E7 mRNA are detected in squamous cell carcinoma and adenocarcinoma of the lung

To provide an accurate evaluation of the association of human papillomavirus (HPV) with lung cancer, 36 cases of lung cancer were analysed for HPV DNAs by polymerase chain reaction (PCR) with dot-blot and Southern blot analyses, and for the transcripts from the E6-E7 transforming region by in situ hybridisation (ISH). HPV-18 DNA was detected in three (8%) of 36 specimens; histologically, in one (10%) of 10 squamous cell carcinomas and two (9%) of 22 adenocarcinomas. Neither HPV-16 nor -33 DNA was detected in any cases examined. Expression of E6-E7 mRNA was confirmed in the cases which contained, HPV-18 DNA. HPV-18 may play an important role in the development and progression of cancer in some cases of both squamous cell carcinoma and adenocarcinoma of the lung.

Expression of E1AF/PEA3, an Ets-related transcription factor in human non-small-cell lung cancers : its relevance in cell motility and invasion

Cell invasion and metastasis characterize the malignant potential of non‐small‐cell lung cancers (NSCLCs). We have previously reported that E1AF, a member of the Ets‐related transcription factor family, confers invasive phenotype on breast cancer and oral squamous‐cell carcinoma cell lines. In our study, we analyzed the E1AF expression in cell lines and resected tumors of NSCLCs by Northern blot and in situ hybridization analyses and found that 15 of 17 cell lines and 12 of 19 tumors expressed E1AF mRNA while normal lung tissue and concomitant normal cells within tumors did not. To examine the biologic importance of E1AF in NSCLCs, we introduced the E1AF gene into VMRC‐LCD and NCI‐H226, NSCLC cell lines lacking E1AF expression, and examined cell motility and invasion activities. E1AF‐transfected VMRC‐LCD cells showed increased cell motility that was 2‐fold that of parental and vector‐transfected control cells (p < 0.01), and both cell motility and invasion were increased 1.6‐fold in NCI‐H226 (p < 0.01). Furthermore, hepatocyte growth factor (HGF), which is one of the most effective cell‐scattering factors, stimulated the motile and invasive activities in E1AF‐transfected VMRC‐LCD and NCI‐H226 cells but not in their parental or vector‐transfected control cells. Ets‐1 mRNA expression was found in E1AF‐transfected VMRC‐LCD cells but not in parental or vector‐transfected cells. HGF further induced expression of the Ets‐1 and urokinase‐type plasminogen activator (uPA) genes specifically in E1AF‐transfected cells. These findings suggest that E1AF plays a substantial role in the cell motility and invasion of NSCLCs.

Increased E1AF expression in mouse fibrosarcoma promotes metastasis through induction of MT1-MMP expression.

In this study, we investigated the role of E1AF, a member of ets family transcription factor, in the acquisition of metastatic capacity by non-metastatic mouse fibrosarcoma cell clone, QR-32. The QR-32 cell clone grows progressively after co-implantation with gelatin sponge in syngeneic C57BL/6 mice. The cell lines (QRsP) established from arising tumors after the co-implantation exhibited enhanced tumorigenicity and pulmonary metastasis in vivo as compared with parent QR-32 cells. The enhanced pulmonary metastasis of QRsP cells was correlated well with augmented production of matrix metalloproteinase-2 (MMP-2) and increased expression of membrane-type 1-MMP (MT1-MMP). The QRsP cells also acquired higher chemokinetic activities to fibronectin and higher invasive activities through a reconstituted basement membrane. Furthermore we observed the elevated mRNA expression of E1AF in QRsP cells compared to parent QR-32 cells. Therefore, we transfected QR-32 cells with E1AF cDNA. Overexpression of E1AF in the QR-32 cells resulted in the induction of MT1-MMP expression and converting an exogenously added precursor MMP-2 into active form. E1AF transfectants exhibited more motile and invasive activities, and moderately increased pulmonary metastatic activities than parental QR-32 cells in vivo, although their metastatic activities were lower than those of QRsP cells. These findings suggest that the increased expression of E1AF in fibrosarcoma contributes to invasive phenotypes including MT1-MMP expression and enhanced cell migration, but not sufficient for exhibiting highly metastatic activity in vivo.