Establishment and characterization of 12 uterine cervical-carcinoma cell lines: Common sequence variation in theE7 gene of HPV-16-positive cell lines

Abstract

Carcinoma of the uterine cervix is one of the most common neoplasms in women, and about 90% of all cervical carcinomas are epidemiologically associated with infection by high-risk human papillomaviruses (HPV) such as types 16, 18, 31 and 33 (Zur Hausen, 1991).Because most available cervical-cancer cell lines were initiated more than 20 years ago (Spence et al., 1988), they have been extensively passaged in culture for hundreds of times in uncontrolled growth conditions, and this might have resulted in changes in the original characteristics of the primary tumor (Spence et al., 1988;Braun et al., 1993). Moreover, because establishment is difficult, the numbers of recently established cell lines with a low passage number are limited (Spence et al., 1988;Braun et al., 1993). The establishment from histopathologically varied primary tumors of cervical-cancer cell lines with a low passage number and an understanding of their characteristics are therefore required.In cervical cancers, a high-risk viral DNA genome is often found to be integrated into the host chromosome by common disruption of the viral E1/E2 region (Schwarz et al., 1985;Chen et al., 1994). In in vitro experiments, E2 repress transcription from the promoters of the E6 and E7 genes. In contrast, E6/E7 genes are actively transcribed in E2-deleted cervical cancers and cell lines (Schwarz et al., 1985).E6 and E7 proteins have been shown to form complexes with the normal cellular protein, p53, or the retinoblastoma-susceptibility gene product, pRb, both of which are well-known tumor suppressors, and those complexes lead to inactivation of the suppression function . Oncogenic HPV-type E6 protein specifically binds to wild-type p53 protein and subsequently enhances the degradation of p53 protein in an ATP/ubiquitin-dependent manner (Scheffner et al., 1990). In addition, pRb is inactivated by binding with E7 protein or through mutation. E7 protein of high-risk HPVs complex with pRb to inactivate the tumor-suppressor function of pRb (reviewed by McIntyre et al., 1993). Functional relevance of alterations at the Rb-binding domain in the E7 gene remains unclear.In this context, we established 12 new uterine cervical-cancer cell lines (SNU-17, SNU-487, SNU-523, SNU-682, SNU-703, SNU-778, SNU-902, SNU-1000, SNU-1005, SNU-1160, SNU-1245 and SNU-1299) with early passages and described cell phenotypes, including histopathology of the primary tumor and in vitro growth characteristics. We also performed molecular characterization, including DNA-fingerprinting analysis and HPV detection, typing and determination of physical form (integrated or episomal) of viral DNA. RNA expression of HPV E6/E7 viral transcripts, sequence variation in the E7 gene of HPV and genetic alteration of p53 were also examined. MATERIAL AND METHODS Cell-line establishment and maintenanceCell lines were established from pathologically proven uterine cervical carcinomas (Table I). Solid tumors were finely minced with scissors and dissociated into small aggregates by pipetting. Appropriate amounts...

Establishment and characterization of four human pancreatic carcinoma cell lines

“…Most of these cell lines were isolated more than 10 years ago, however, and have been passaged many times in vitro in uncontrolled growth conditions. Therefore, they could have lost their phenotypic heterogeneity, like cervical cancer cell lines commonly observed in primary tumor lines (reviewed by Ku et al, 1997). The use of pancreatic carcinoma cell lines with a low passage number would solve this problem.…”

The molecular genetics of cervical carcinoma

“…In another study, alterations at eight dinucleotide loci mapping to six different chromosomes were found in five out of 82 cervical cancers (Helland et al, 1997;Kersemaekers et al, 1998a). In another 51 cases, mutator phenotypes were detected in four cases (Ku J-L et al, 1977;Ku W-H et al, 1997). This instability affected to one or two alleles at one or few loci.…”

“…Human Yokota et al, 1989;Jones and Nakamura, 1992;Kohno et al, 1993;Karlsen et al, 1994;Mitra et al, 1994a;Mullokandov et al, 1996;Ku et al, 1997;Larson et al, 1997;Chu et al, 1998;Huettner et al, 1998;Kersemaekers et al, 1998b 4p16 27/66 40 Hampton et al, 1996;Mullokandov et al, 1996;Kersemaekers et al, 1998b 4q21-35 23/72 32 Mitra et al, 1994a;Mullokandov et al, 1996;Kersemaekers et al, 1998b 5p13-15 20/117 17 Ku et al, 1977;Mitra et al, 1994a;Mullokandov et al, 1996;Kersemaekers et al, 1998b 6p21.3-22 60 keratinocytes immortalized with HPV-16 DNA show LOH at 3p. The tumorigenicity of these cells in nude animals correlated very well with instability and loss of genetic material from human chromosome 3p (Montgomery et al, 1995;Steenbergen et al, 1996), 10p, 11q and 18q regions (Montgomery et al, 1995;Steenbergen et al, 1995Steenbergen et al, , 1996.…”

“…Overall, of 344 cases analysed for the presence of the mutator phenotype, only 28 displayed MI (8%), independently of the type of allele and number of chromosomes studied (Ku J-L et al, 1977;Larson et al, 1996;Helland, 1997;Ku W-H et al, 1997), which represents a small subset of the total number of CC cases.…”

Establishment and characterization of seven human renal cell carcinoma cell lines

“…Appropriate amounts of ®ne neoplastic tissue fragments were seeded into 25-cm 2¯a sks. Tumour cells were initially cultured in AR5 medium, which has proved useful in establishing human cancer cell lines [18,19] and the composition of which was described previously [18]. After being established, the cultures were maintained in RPMI 1640 (Gibco/BRL, Grand Island, NY) supplemented with 10% heatinactivated fetal bovine serum.…”