Bharati Hukku

Cell Characterization by Use of Multiple Genetic Markers

The extensive use of cell cultures for diverse research purposes is one of the truly great international growth industries. With the proliferation of cells comes a responsibility for monitoring them for inter- and intraspecies characteristics. We use multiple genetic markers for cell identification, i.e. species specific antigens, isozymic phenotypes, chromosomal complement, and HL-A haplotypes. The methodologies employed are briefly described, and various examples cited to show how these markers can be utilized for cell line monitoring. Data are summarized from 275 cultures sent to our laboratory for analysis during the past eighteen months. The data show that, overall, 35% of the cultures received were contaminated. The majority of cell cultures submitted were human cell lines. We found that 36% of these cultures were cross contaminated; 25% by cells of another species and 11% by another human cell line. This high incidence of inter- and intraspecies contamination underscores the importance of frequent monitoring of cell cultures.

Gene Amplification and Increased Expression of the Reduced Folate Carrier in Transport Elevated K562 Cells

The molecular bases for the 6-fold elevated methotrexate transport capacity of K562.4CF cells (Matherly et al., Cancer Res. 51: 3420-3426, 1991) were studied with reduced folate carrier (RFC) cDNA, genomic, and antibody probes. Southern analysis showed that RFC gene copies were increased (approximately 4- to 5-fold) in K562.4CF over wild-type K562 cells. Fluorescence in situ hybridization using a genomic RFC probe confirmed the localization of the RFC gene to the q-arm of chromosome 21. In K562.4CF cells, the frequent loss of a normal copy of chromosome 21 (61% of metaphases) was accompanied by RFC gene amplification and translocations of amplified RFC gene fragments to several (2 to 6) different chromosomal loci not seen in wild-type cells. Particularly intense RFC signals were mapped to homogeneously staining regions in chromosomes 2 and 15. Increased RFC gene copies were accompanied by a similar increase in the major 3.1 kb RFC transcript by northern blotting and an approximately 7-fold elevated level of the broadly migrating (80-95 kDa) RFC protein on a western blot probed with an RFC C-terminal peptide antibody. These results demonstrate that selection of cells with a growth-limiting concentration of reduced folates (0.4 nM of leucovorin) is sufficient to promote chromosomal aberrations, including gene amplification and translocations that result in increased RFC expression and folate transport.

Role of chromosome 5 in immortalization and tumorigenesis of human keratinocytes.

Rhim et al. were first to show that superinfection of Ad12-SV40-infected immortalized human epidermal cells with an RNA tumor virus containing a ras oncogene, such as Ki-MSV, or their treatment with chemical carcinogens, leads to the ability of cells to both grow in anchorage-independent fashion and to form tumors in athymic nude mice. We describe details of the chromosome changes observed during the transformation. The culture was monitored through 40 passages after Ad12-SV40 infection. Chromosomes 9 and 11 showed random monosomy during the initial stages, but by passage 10 clonal evolution of the cell line was well established. Observed chromosome monosomy/trisomy coupled with chromosome rearrangements (identified as chromosomes A through F) were monosomy 13, loss of p arms of 8 and 10, partial loss of 5 (del(5)(q13) and of the q arm of 18(del(18)(q12)), and extra copies of 11q, 20 and 21. During its progression to tumorigenicity, a derived chromosome E containing a segment of 5q, also appeared to play a major role. The cells remained immortalized as long as the 5q segment was present in some of the cell population as derived chromosomes E or F. Derivative chromosome E showed noteworthy changes during the progression to tumorigenicity, in both viral and chemical transformations. There was loss of heterozygosity of 5q due to an exchange of 5q with chromosomes E or F. In Ki-MSV- and 4NQO-transformed cells, presence of an altered chromosome E (identified as E1) was observed. In MNNG-treated cells, there was a selection of population of cells with further alteration in chromosome E (identified as E3). Besides alterations in chromosome E, additional chromosome changes leading to gene activation and amplification indicating a multistep progression to tumorigenicity were observed. The cytogenetic data reiterate the ever-increasing need for molecular analysis of nonrandom karyotype changes.

Stepwise genetic changes associated with progression of nontumorigenic HPV-18 immortalized human prostate cancer-derived cell line to a malignant phenotype

Cytogenetics, fluorescence in situ hybridization (FISH), and comparative genomic hybridization (CGH) were used to identify genes that are involved in the development and progression of prostate cancer. For that purpose, we chose a cell line established in vitro from a prostatic adenocarcinoma which was nontumorigenic in nude mice and followed its progression to a tumorigenic cell line. Stepwise changes were observed in the cell line as it became tumorigenic. The composite karyotype at the nontumorigenic stage (CA-HPV-10) was 68 approximately 77,XXY,-(1, 9, 13, 14, 19, 22),+(4, 5, 11, 18, 20, 21),+(del(1) (q23q31)=M1 (two copies), +der(9)t(1;9)(q24 approximately q31;p23)=M5(two copies), der(14)t(14;?)(q10;?)=M17 in the majority of metaphases. These two derivative chromosomes were also observed a previous study. Our CGH analysis clearly showed that this deleted region in M1 is, in fact, translocated with derivative M5 and, in reality, is amplified. The cell line established from nodule (SCID 5019 p11), showed a number of new changes, as described; however, the most significant change was amplification of the 8q23 approximately qter region, harboring c-myc. This region was translocated with chromosomes 2, 4, and 16 as der(2)t(2;8)(q33;q23)=M12, der(4)t(4;8)(q34;q23)=M11, and der(16)t(8;16)(q24;q21)=M9. We deduce from our study that amplification of c-myc and other genes in the 8q23 approximately qter region were important in progression but did not lead to tumorigenicity. The population that became tumorigenic (SCID 5019 II) showed almost all of the same changes in the karyotype as observed in the nodular cell line; the only significant change was the appearance of der(11)t(4;11)(q32;q22)=M7 and the addition of another copy of t(3q;7p)=M2. These new changes lead to loss of chromosomes 3p, 4pter approximately q34, 6, 7q21 approximately qter, 11q22 approximately qter, and 18q, and gain of 3q, 7p, 8q23 approximately qter, and 11pter approximately q22, before the cell line became tumorigenic. The clonal selection of the population is proven by the presence of a number of the same derivative chromosomes in both the nodular and tumorigenic cell line. As it progressed to tumorigenicity, some of the same changes observed in the original study re-appear at different stages of malignancy, although it was absent in the nontumorigenic cell line. These are: der(16)t(8;16)(q24;q21)=M9 in the nodular cell line and der(11)t(4;11)(q32;q22)=M7 in the tumorigenic cell line. In our system, amplification of c-myc and other genes in der(2)t(2;8)(q33;q23)=M12,der(4) t(4;8)(q34;q23)=M11 together with the presence of der(16)t(8;16)(q24;q21)=M9 and der(11)t(4;11)(q32;q22)=M5 makes the cell line tumorigenic. It is either nontumorigenic, with the presence of a marker equivalent to der(16)=M9 and der(11)=M7 observed in the original study, and only nodular (SCID 5019 p11, present study), with the presence of number of markers with c-myc amplification (M9, M11, and M12). There is accumulation of all the above-mentioned changes in the same cell before it becomes tumorigenic.

CELL LINE CHARACTERIZATION AND AUTHENTICATION

Research and development involving the use of cell lines require precise knowledge of the purity and species of origin of the cell lines used. This can only be assured by periodic monitoring of cultured cell lines for possible contamination by other cells and for characteristics that authenticate the cell line identity. In the absence of such monitoring, inter- and intraspecies cell line contaminations are likely to occur in the laboratories of unsuspecting investigators and can result in the generation of mistaken conclusions with an attendant loss of investigators time, effort, and resources. This chapter provides a history and an overview of the methods that have been developed for cell line authentication, the type of information each of these different methods provides, and how synthesis of that information can be used to characterize a cell line and confirm its identity. An effective cell line monitoring strategy is described that involves testing for a combination of genetic markers, including cell membrane species antigens, isoenzymes, chromosomes, and DNA fingerprints, and use of databases for each marker system to compare the results obtained with a test cell culture with results from an extensive panel of previously tested cell lines.

Chromosomal changes observed in immortalized human keratinocytes transformed by ionizing radiation.

Human epithelial cancer cells were induced by concerted action of DNA tumor virus and X-ray radiation. Treatment of nontumorigenic early passage AD12-SV40 immortalized epithelial cells (RHEK-1) at passage 23 with radiation, resulted in further changes in their growth properties. One day old cultures of these RHEK-1 cells were irradiated with graded doses of X-rays (0, 2, 4, 6, and 8 Gy i.e. RHEK-1, RHEK-1/200R, RHEK-1/400R, RHEK-1/600R, and RHEK-1/800R). Morphologic alterations, the ability to grow in soft agar, and to form rapidly-growing squamous cell carcinomas in nude mice were concomitantly acquired properties of the radiation transformed cell lines RHEK-1/200R and RHEK-1/ 400R. On the basis of commonality in having addition of some extra material in chromosome 11 in the region between q14/q22 in all tumorigenic cell lines RHEK-1/200R and RHEK-1/400R, and deletion of the same region in nontumorigenic irradiated cell lines-RHEK-1/600R and RHEK-1/800R, it is deduced this region may have some important oncogene/s or other gene/s that play an important role in tumorigenesis. When compared to squamous cell carcinoma data, the duplication observed in the present study is also observed in 28 to 38% of head and neck and also in 25% of cases of untreated malignant lesions of oral squamous cell carcinoma. Thus, this study shows the correlation between in vitro induced squamous cell carcinoma to in vivo tumors.

Development of a new human breast cancer cell line Ia-270.

SummaryA new human breast cancer cell line (Ia-270) has been isolated from a malignant pleural effusion from a woman with metastatic infiltrating ductal carcinoma of the breast. This cell line contains cytoplasmic estrogen (ER) and progesterone (PR) receptors. Following estradiol (E2) administration, PR synthesis is augmented and a higher level of saturation density is reached. In an athymic mouse, the cell line produced a tumor morphologically similar to the primary tumor. The results of isoenzyme and karyotype analyses demonstrate Ia-270 to be of human origin and free of HeLa cell contamination. The cell line has been maintained in continuous culture since April 1982 and may provide a usefulin vitro system for studying the biology of human breast cancer.

A NEW CELL LINE EXPRESSING A NOVEL TYPE OF TMPRSS2- ERG GENE FUSION DERIVED FROM PRIMARY TUMORS OF FAMILIAL PROSTATE CANCER PATIENT

194 Recent studies have established that a high proportion of prostate cancer harbors a gene fusion between the androgen regulated TMPRSS2 gene and the ETS genes ERG, ETV1 or ETV4. However in vitro model representing primary tumors to study the molecular mechanisms and functional consequences of this important chromosomal rearrangement are currently limited and are greatly needed. The tumor tissue (RC-123T) used for generating the RC-123T/E cell line with HPV-16 E6E7 genes was obtained from a 57 year old familial prostate cancer patient who had adenocarcinoma with well differentiation (Gleason 3+3). The cell line was characterized phenotypically and cytogenically. Dual color fluorescence in situ hybridization (FISH) assay was used to test for ERG (3’ 5’) break aparts, and for translocation of TMPRSS2 and ERG and ETV1, in both interphase nuclei as well as metaphases. The cells were also characterized for putative stem cell markers CD133, SOX2 and prostate cell markers by immunohistochemistry and RT-PCR. We have successfully established an immortalized human prostate epithelial cell culture derived from primary tumors of familial prostate cancer patients carrying TMPRSS2-ERG fusion genes with HPV16 E6E7 genes (RC-123T/E). RC-123T/E cells are currently growing well at passage 30, whereas RC-123T cells senesced at passage 5. Expression of an androgen-regulated prostate specific homeobox gene, NKX3.1, the epithelial cell-specific cytokeratin 8 and androgen receptor but not prostate specific antigen, was detected in RC-123T/E cells. TMPRSS2-ERG genes were also expressed in this line. The RC-123T/E cells formed spheres under suspension culture and branched differentiated in matrigel. The RC-123T/E cells also expressed putative stem cell markers CD133 and SOX2. Interestingly, the FISH analysis showed the translocation of chromosome t(7;21)(q21:q21) in the RC-123T/E cell line and also in its primary tumor tissue. We demonstrate that RC-123T/E cell line and its primary tumor tissue possess the translocation of TMPRSS2/ERG to chromosome 7q which adds another class of gene arrangement in prostate cancer. In addition, this cell line expressed putative stem cell markers and TMPRSS2-ERG gene fusion. This model provides a novel tool to study the cellular and molecular mechanisms of TMPRSS2-ETS genes in prostate cancer.