Tumorigenicity of human hematopoietic cell lines in athymic nude mice.

Human hematopoietic cell lines, which had been classified on the basis of studies on clonality, and morphological, chromosomal and functional parameters as lymphoblastoid cell lines (LCL) of presumed non-neoplastic origin, and lymphoma, myeloma and leukemia lines of proven malignant origin, were tested for tumorigenic potential on subcutaneous transplantation to nude mice and for capacity to grow in semi-solid medium in vitro. Recently established LCL failed to grow both in nude mice and in agarose. In contrast, some of the LCL which had developed secondary chromosomal alterations during continuous cultivation for periods exceeding several years were tumorigenic and/or had the capacity to form colonies in agarose. Most lymphoma lines formed colonies in agarose and tumors in the mice. One of the two myeloma lines formed subcutaneous tumor which, however, showed no progressive growth. The other myeloma line failed to grow. Both myeloma lines, however, formed colonies in agarose. The myeloid leukemia line was tumorigenic while two of the three tested lymphocytic leukemia lines failed to grow in the mice. All leukemia lines formed colonies in agarose. We conclude from this study that: (1) Of the two types of Epstein-Barr virus containing cell lines [LCL and Burkitt lymphoma (BL) lines], only BL lines were shown to form tumors when inoculated subcutaneously in nude mice and had the capacity to grow in agarose in vitro. This shows that EBV transformation per se does not necessarily render lymphocytes tumorigenic in nude mice. The capacity to form colonies in agarose is not acquired either. (2) Changes of the karyotype and several phenotypic characteristics which occur in the originally diploid LCL during prolonged cultivation in vitro may be accompanied by the acquisition of the potential to grow subcutaneously in nude mice and in agarose in vitro. (3) The inconsistency with regard to the capacity of come of the neoplastic cell lines to grow in nude mice or in agarose seems to underline that neither of the two tests is a reliable criterion for malignancy of human lymphoma, leukemia and myeloma cell lines.     

Growth in culture and tumorigenicity after transfection with the ras oncogene of liver epithelial cells from carcinogen-treated rats

Two epithelial cell lines designated LE/2 and LE/6 were established from cells isolated by centrifugal elutriation from the livers of carcinogen-treated rats. Both cell lines exhibit some characteristics of fetal liver cells, such as the expression of the 2.3-kilobase alpha-fetoprotein mRNA, aldolase A, and lactate dehydrogenases 4 and 5. Primary cultures contain gamma-glutamyl transferase-positive cells which do not proliferate in vitro. After the first passage, the LE/2 and LE/6 cell lines are uniformly gamma-glutamyl transferase negative. Neither cell line is transformed as assayed by morphology, anchorage-independent growth, or tumor formation in nude mice. By the 50th passage, LE/6 cells form numerous colonies in soft agar in the presence of epidermal growth factor, while no colonies grow in medium lacking this growth factor. Clonal cell populations derived from five epidermal growth factor-induced soft agar colonies were not tumorigenic in nude mice. This indicates that, although epidermal growth factor-responsive late passage cells had acquired some of the phenotypic properties commonly associated with tumor cells, these cells were not fully transformed. Transformation of LE/6 cells was accomplished by transfection of the rasH oncogene (EJ). Subcutaneous inoculation of rasH (EJ)-transfected LE/6 cells produced tumors at the site of injection with histological features of moderate to well-differentiated trabecular hepatocellular carcinomas. Tumor cell lines derived from the nude mouse tumors are gamma-glutamyl transferase positive and express alpha-fetoprotein mRNA. One clonal cell line expresses both alpha-fetoprotein and albumin mRNA. These results show that nonparenchymal liver epithelial cells transfected with an activated oncogene can give rise to differentiated hepatocellular tumors similar to those induced in livers of rats fed a carcinogenic diet.     

Reconstituted basement membrane (matrigel) enhances the growth of human glioma cell lines in nude mice

Transplantation of human cancers into immunologically deficient mice is widely used to study potential therapeutic interventions in vivo. For brain tumor research, however, several factors limit more widespread application of this animal model. First, only a minority of human glioma-derived cell lines are tumorigenic in nude mice. In addition, even for tumorigenic cell lines, tumor take is variable and growth is often slow for tumors derived from cell inoculums. Reconstituted components of tumor basement membrane (matrigel) have been found to improve the growth in nude mice of several types of human tumors originating outside the central nervous system when premixed with the tumor cells before subcutaneous inoculation. We investigated the ability of matrigel to enhance the growth in nude mice of tumors derived from the human glioma cell lines U-251 MG, U-373 MG, SNB-78 and SNB-101.Athymic nude mice (NIH Swiss background, nu/nu genotype) were inoculated subcutaneously with 1.0 × 10⁶ tumor cells alone or after premixing with an equal volume of liquid matrigel. U-251 and U-373 cells were tumorigenic, with palpable tumors present by about 2 to 3 weeks. Co-injection of these cell lines with matrigel resulted in higher tumor-take rates, from 6/10 to 8/8 animals for U-251 at 60 days, and from 9/12 to 11/11 animals for U-373 at 60 days. Matrigel also enhanced tumor growth, with tumors at 45 days significantly larger than those formed in the absence of matrigel, for both cell lines (p < 0.01). SNB-78 and SNB-101 cells did not give rise to progressively enlarging solid tumors with or without matrigel.Matrigel enhances the growth of tumorigenic human gliomas in athymic nude mice. This technique provides a model with more consistent tumor take and more rapid growth kinetics for human glioma cell lines that are tumorigenic in nude mice.The U.S. Government right to retain a non-exclusive, royalty free licence in and to any copyright is acknowledged     

Study of nm23 gene expression in an N-methyl-N-nitrosourea transformed human breast epithelial cell line

In the present study we pinpoint the time of nm23 down-regulation during the chemical transformation of the human breast epithelial cell line HBL100. The non-malignant HBL100 was transformed by exposing it to N-methyl-N-nitrosourea (MNU). We subsequently injected the transformed cells (HBL-T-MNU) into nude mice, resulting in tumor growth. With a second passage of these tumors in mice we observed lung and extra-regional node metastases. The expression of nm23 in the non-tumorigenic HBL100 cells was compared to the tumorigenic KBL-T-MNU cells as well as to the metastatic cell line HBL-T2(MNU) derived from the tumor induced by HBL-T-MNU cells. By using immunohistochemistry and Western blot analysis we documented the downregulation of nm23 expression in the tumorigenic HBL-T-MNU and metastatic HBL-T2(MNU) cell lines, as compared to the parental line HBL100. Once downregulated, nm23 expression in this model remains constant during the subsequent progression. These results suggest that nm23 down-regulation may indeed be associated with early neoplastic transformation and is maintained throughout neoplastic progression and metastatic stage.     

Establishment and characterization of a continuous lung squamous cell carcinoma cell line (U-1752)

A continuous cell line, U-1752, was established from a lung tumor originally diagnosed as a small cell carcinoma. The cell line has been in continuous in vitro passage for 29 months. The epithelial, rather than small cell nature of the U-1752 cells was demonstrated by the presence of desmosomes, prominent tonofilament bundles, by the reactivity with an anti-keratin antiserum and by the expression of cell surface receptors for epidermal growth factor (EGF). The U-1752 cells grow as monolayer cultures and have a population doubling time of around 36 hours at optimal growth in 20% calf serum. The most important neoplastic features of U-1752 were its aneuploidy, its capacity for colony formation in agarose and its tumorigenic potential subcutaneously in nude mice.     

Growth of Hodgkin cell lines in severely combined immunodeficient mice.

No animal model exists for the in vivo growth of Hodgkin's-lymphoma-derived cells. Neither unmanipulated Hodgkin's-disease(HD)-derived cell lines nor primary biopsy tissue could be grown in nude mice. Since the severe combined immunodeficient (SCID) mouse has been reported to be a better recipient for transplanted human lymphatic tissue than the nude mouse, we tested whether SCID mice provide suitable conditions for the in vivo growth of HD cell lines. Tumorigenicity of HD cells was tested in untreated and pre-treated SCID mice and in another combined immunodeficient mouse strain, beige/nude/X-linked immunodeficient (BNX) mouse. SCID mice supported in vivo growth of the 6 HD cell lines tested (L428, L540, L591, DEV, HD-LM2, KM-H2). Only one of the 6 lines (DEV) was tumorigenic in BNX mice. No HD cell line proliferated in T-cell-deficient nude mice. Thus, in vivo growth of HD cell lines appeared to be related to the degree of host immunodeficiency. Additional growth supportive treatments such as fibrosarcoma co-transplantation, intraperitoneal mineral oil injection or immunosuppressive pre-treatment (anti-asialo-GMI-antibody injection) permitted growth of 3 additional HD cell lines in BNX mice. The immunophenotype and karyotype of explanted graft cells were identical to the original cell lines. Our experiments describe an effective and reproducible xenograft model for growth of Hodgkin's-disease-derived cell lines. This may be of value for elucidating the growth characteristics of Hodgkin's-lymphoma-derived cells as well as for testing new therapeutic regimens.     

Differential effects of selenium on the growth of mouse mammary cells in vitro

The effect of selenium was examined on the growth potential of normal, preneoplastic and neoplastic mammary cells grown in primary monolayer cell cultures and on 3 established mammary cell lines. Selenium, present as Na₂SeO₃ in serum-free DMEM, inhibited all mammary cell cultures at 1 × 10^?5 M. Selenium, at 5 × 10^?8 M, stimulated the growth of primary cell cultures of normal mammary cells and C4 preneoplastic cells and the established cell line YN-4, but not the growth of D2 preneoplastic cells and tumors in primary cell cultures and of established cell line CL-S1 and WAZ-2t. The differential responses of cells from preneoplastic outgrowth lines C4 and D2 and of D2 primary tumors in vitro correlated with the sensitivity of these same cell populations to selenium-mediated inhibition of growth and tumorigenesis in vivo. The differential responses among the 3 cell lines will allow further comparative studies on the cell biological basis of selenium function.     

Intracranial heterotransplantation of human hematopoietic cells in nude mice.

A panel of established cell lines and many primary cell specimens from lymphomas and leukemias as well as from normal lymphatic tissues were tested for tumorigenicity by intracranial heterotransplantation in nude mice. Not only lymphoma and leukemia cell lines, but also lymphoblastoid cell lines, lacking markers of malignancy, were tumorigenic in the brains of nude mice. These findings indicate that tumorigenicity following intracranial heterotransplantation in nude mice cannot be used as proof for the malignant nature of established cell lines. Heterotraplantation of primary cell specimens yielded only a few tumor takes. When primary cells were infected with exogenous Epstein-Barr virus prior to the transplantation procedure, tumorigenicity could be significantly increased. Cytogenetic evaluation of tumors growing after intracranial transplantation of human hematopoietic cells showed, in some cases, a selection of cytogenetically aberrant cell clones.     

Establishment of a human large cell lung tumor line (QU-DB) with metastatic properties in athymic mice

A continuous human cell line was established from a patient with large cell anaplastic lung carcinoma. This cell line, designated QU-DB, has been in culture for over 36 months and grows as an adherent monolayer with a doubling time of 10-12 hours. Its morphology, ultrastructure, karyotype, ability to grow in soft agar and heterotransplantability, indicate it is a large-cell lung tumor cell line of human origin. Three cell lines were established from metastatic tumors in nude mice receiving subcutaneous injections of QU-DB cells. The morphology and growth characteristics exhibited by these cell lines were similar to the primary cell line. Karyotypic analysis of cell lines derived from the primary tumor and a metastasis to the diaphragm were similar, but cells from a liver metastasis culture showed additional karyotypic changes. This large cell lung tumor cell line may prove useful as a model system for studies of human tumor progression and metastasis.     

Growth and metastasis of tumor cells isolated from a human renal cell carcinoma implanted into different organs of nude mice

The purpose of this study was to determine whether the methods for isolating tumor cells from a human renal cell carcinoma (HRCC) influence the biological behavior of the cancer cells. Renal cell carcinoma obtained from a surgical specimen was dissociated by enzymatic treatment and cells were plated into culture dishes or injected s.c. into the kidney of BALB/c nude mice. The resultant kidney tumor produced liver metastasis and ascites. All tumors growing in nude mice (s.c., kidney, liver, ascites) were also established in culture. The human origin of all five lines was ascertained by karyotypic and isoenzyme analyses. Cells from all lines were injected, s.c., i.p., i.v., intrasplenically, and beneath the renal capsule of nude mice. All the lines were tumorigenic after s.c. or renal subcapsule injection, although the rate of tumor growth varied among the five lines. The metastatic behavior of the HRCC cells was influenced by both the nature of the tumor cells and the route of injection into nude mice. In general, cells derived from the liver metastasis produced more metastases in nude mice than other lines. The lines established in culture from the primary HRCC and the ascites were poorly metastatic. Even with highly metastatic cells, i.v. injection did not yield significant metastasis, but the injection of cells into the renal subcapsule resulted in extensive metastasis to the lungs and in all peritoneal organs. These results indicate that nude mice can be used for the isolation of populations of HRCC cells with different growth and metastatic potential and that, of the organ sites tested, the renal subcapsule is the most advantageous site for implantation of HRCC cells.     

Androgen responsive adult human prostatic epithelial cell lines immortalized by human papillomavirus 18

Prostate cancer and benign tumors of the prostate are the two most common neoplastic diseases in men in the United States, however, research on their causes and treatment has been slow because of the difficulty in obtaining fresh samples of human tissue and a lack of well characterized cell lines which exhibit growth and differentiation characteristics of normal prostatic epithelium. Non-neoplastic adult human prostatic epithelial cells from a white male donor were immortalized with human papillomavirus 18 which resulted in the establishment of the RWPE-1 cell line. Cells from the RWPE-1 cell line were further transformed by v-Ki-ras to establish the RWPE-2 cell line. The objectives of this study were to: (1) establish the prostatic epithelial origin and androgen responsiveness of RWPE-1 and RWPE-2 cell lines; (2) examine their response to growth factors; and (3) establish the malignant characteristics of the RWPE-2 cell line. Immunoperoxidase staining showed that both RWPE-1 and RWPE-2 cells express cytokeratins 8 and 18, which are characteristic of luminal prostatic epithelial cells, but they also coexpress basal cell cytokeratins. These cell lines show growth stimulation and prostate specific antigen (PSA) and androgen receptor (AR) expression in response to the synthetic androgen mibolerone, which establishes their prostatic epithelial origin. Both cell lines also show a dose-dependent growth stimulation by EGF and bFGF and growth inhibition when exposed to TGF-beta, however, the transformed RWPE-2 cells are less responsive. RWPE-1 cells neither grow in agar nor form tumors when injected into nude mice with or without Matrigel. However, RWPE-2 cells form colonies in agar and tumors in nude mice. In the in vitro invasion assay, RWPE-1 cells are not invasive whereas RWPE-2 cells are invasive. Nuclear expression of p53 and Rb proteins was heterogeneous but detectable by immunostaining in both cell lines. The RWPE-1 cells, which show many normal cell characteristics, and the malignant RWPE-2 cells, provide useful cell culture models for studies on prostate growth regulation and carcinogenesis.      

Establishment and characterization of human renal cancer and normal kidney cell lines

We have reviewed our laboratory's efforts to establish continuous human renal cancer cell lines. During the 16-year period of 1972 through 1987, 498 successive attempts resulted in establishment of 63 renal cancer cell lines. Of these lines, 46 were derived from primary kidney tumors and 17 from metastatic sites (lung, brain, bone, and lymph node). Forty-three of these lines have been characterized with regard to morphology, growth kinetics, anchorage-independent growth, tumorigenicity in athymic nude mice, and expression of kidney cell surface antigens. These results were compared with data from primary short term cultures of normal kidney epithelium. The overall success rate of establishing continuous renal cancer cell lines was 12.7%. In general, no significant difference in success was noted based on whether the specimen was derived from a primary or a metastatic lesion. However, all successfully established lines were derived from tumors exhibiting clinically "aggressive" behavior. All cell lines expressed proximal tubular cell differentiation antigens. Significant morphological heterogeneity was observed among normal kidney as well as kidney cancer cell lines in vitro. No significant difference in doubling time was found between cell lines of renal cancer and passage 1 cultures of normal kidney epithelium. Twenty-one of 30 (70%) lines assayed formed clones on soft agar and 26 of 33 (79%) lines grew in athymic mice. Among the 25 lines which were assayed for both soft agar growth and tumorigenicity in nude mice, this pair of phenotypic traits were concordant in 17 lines (60%). Four lines (16%) grew on agar but not in mice, while four other lines (16%) failed to grow in agar but were tumorigenic in mice.     

Transformation by inorganic arsenic compounds of normal Syrian hamster embryo cells into a neoplastic state in which they become anchorage-independent and cause tumors in newborn hamsters

Arsenic is a known human carcinogen, but little evidence exists for its carcinogenicity in animals. In order to investigate the ability of inorganic arsenics to transform normal cells into a neoplastic state, mass cultures of normal, diploid Syrian hamster embryo (SHE) cells exposed to various concentrations of sodium arsenite or sodium arsenate for 48 hr were continually passaged and tested for neoplastic transformation, as determined by anchorage-independent growth in semisolid agar and tumorigenicity in newborn hamsters. Twenty-one of 22 (96%) untreated, control cultures senesced by 20 passages. While 1 culture escaped senescence, it did not acquire the ability to either grow in semisolid agar or form tumors in animals. Ten of 14 (71%) cultures exposed to sodium arsenite or sodium arsenate escaped senescence. Nine of the 10 (90%) arsenic-treated immortal cultures acquired the anchorage-independent phenotype. Five of 5 anchorage-independent cultures examined were tumorigenic. Two of 3 morphologically transformed colonies induced by sodium arsenate also acquired the ability to grow in semisolid agar when isolated. Amplification of the c-myc or c-Ha-ras oncogene was detected in 3 of 5 and 4 of 5 tumorigenic cell lines, respectively. Both c-myc and c-Ha-ras were amplified even in a preneoplastic, anchorage-dependent cell line, but neither was amplified in 6 of 9 anchorage-independent cell lines. Overexpression of c-myc and c-Ha-ras mRNA was observed in most of the neoplastically transformed cell lines but not in the preneoplastic cell line. Experiments using the methylation-sensitive restriction endonuclease isoschizomers HpaII and MspI revealed hypomethylation of c-myc and c-Ha-ras in the 5'-CCGG sequence of arsenic-exposed cell lines but not in the parental SHE cells or a spontaneously transformed cell line. Thus, inorganic arsenics induce neoplastic transformation of normal, diploid mammalian cells. Overexpression of oncogenes by DNA hypomethylation may participate in the arsenic-induced neoplastic transformation of mammalian cells.     

Neoplastic transformation of a human bronchial epithelial cell line by a recombinant retrovirus encoding viral Harvey ras

Activated ras oncogenes have previously been implicated in the pathogenesis of human lung carcinomas. A v-Ha-ras-containing retrovirus, Zip-ras, was generated by inserting the coding region of the v-Ha-ras oncogene into the Zip-NeoSV(X) [Cepko et al., Cell 37:1053-1062, 1984] retroviral vector. Amphotrophic Zip-ras retrovirus was used to infect an SV40 large T antigen-positive immortalized cell line, BEAS-2B, derived from normal bronchial epithelial cells, the predominant progenitor cells of human lung carcinomas. Zip-ras-infected BEAS-2B cells selected for G418 resistance formed anaplastic carcinomas in 12 of 15 athymic nude mice (latency 3 wk), whereas Zip-NeoSV(X)-infected BEAS-2B control cultures inoculated into 12 nude mice formed no tumors after a minimum of 7 mo. Tumor cell lines were established and demonstrated to be of human epithelial origin and to express v-Ha-ras p21 protein. A common feature of the tumor cell lines was an increase in ploidy. The increased efficiency of neoplastic transformation by v-Ha-ras of cell lines as compared with our previous results with normal bronchial epithelial cells [Yoakum et al., Science 227:1174-1179, 1985] is consistent with the hypothesis that the "immortalization" step is rate-limiting in in vitro human epithelial cell carcinogenesis.     

ras transformation of simian virus 40-immortalized rat hepatocytes: an in vitro model of hepatocarcinogenesis.

Primary rat hepatocytes were transfected with simian virus 40 DNA and cultured in a chemically defined medium. Proliferating colonies developed after 2-3 weeks. Three cell lines were established by cloning albumin-secreting colonies, as identified by an immunooverlay assay. Two of the cell lines, ALB-6 and ALB-8, expressed all five liver-specific mRNAs studied, albumin, alpha-1-antitrypsin, fibrinogen, alpha-1-acid glycoprotein, and histidase. ALB-6 cells were nontumorigenic in nude mice while ALB-8 cells were weakly tumorigenic with only one of four injected nude mice developing a slowly growing tumor. Further transfection of ALB-6 and ALB-8 cells with an activated c-Ha-ras or N-ras oncogene resulted in strongly tumorigenic cells. The tumors induced by ras-transformed ALB-6 cells were moderately differentiated hepatocellular carcinomas. The tumors derived from ras-transformed ALB-8 cells were poorly differentiated, while the slowly growing tumors induced by untransfected or control DNA-transfected ALB-8 cells were well-differentiated trabecular hepatocellular carcinomas, suggesting histological dedifferentiation of cells following ras transformation. However, the synthetic capabilities of the cells were not lost in that the ras-transfected cultures and the tumors induced by ras-transformed cells retained the ability to synthesize the five liver-specific mRNAs. Thus we have developed an in vitro model of carcinogenesis in which, by sequential exposure to SV40 DNA and a ras oncogene, primary rat hepatocytes are transformed.     

Differential tumorigenicity of two autologous human breast carcinoma cell lines, HMT-3909S1 and HMT-3909S8, established in serum-free medium

In a serum-free medium we have established two new human breast carcinoma cell lines from a single primary tumor. Cultures were maintained on chemically defined medium CDM3 or on minor modifications of this medium, Dulbecco's modified Eagle medium-Ham's F12 supplemented with epidermal growth factor, insulin, transferrin, estradiol, hydrocortisone, triiodothyronine, cyclic AMP, phosphoethanolamine, ethanolamine, fibronectin, fetuin, ascorbic acid, bovine serum albumin, and trace element salts including selenite (Petersen and van Deurs, Cancer Res., 47: 856-866, 1987). Primary cultures comprised both NADPH-neotetrazolium reductase-positive carcinoma cells and NADPH-neotetrazolium reductase-negative cells of stromal appearance, as well as normal epithelial cells (Petersen and van Deurs, Cancer Res., 46: 2013-2020, 1986). In subsequent passages the cells were monitored exclusively using the tumorigenicity assay on nude mice. Two cell lines, one nontumorigenic, HMT-3909S1, and one tumorigenic, HMT-3909S8, were selected from the primary cultures. Selection of S8 through subline S4 required transient supplementation of CDM3 with fetal calf serum. Permanent lines S1 and S8 were maintained on serum-free medium. Further characterization of the two cell lines in terms of normal breast gland differentiation (Petersen and van Deurs, Differentiation, 39: 197-215, 1988) was carried out using immunocytochemistry, immunochemistry, electron microscopy, and cytogenetics. S8 appeared to be identical with the NADPH-neotetrazolium reductase-positive carcinoma cells of the primary cultures, with a particular subpopulation of carcinoma cells in the tumor of origin, and with the tumorigenic cells of the nude mice. This subline was aneuploid, typically epithelial in morphology, and expressed keratins K8 and K18 and the glycoprotein MAM-6, typical of luminal epithelial cells in the normal breast gland. Subline S1 appeared more like the elongated cells in the primary cultures and like a second subpopulation of cells in the carcinoma of origin. However, S1 cells were in fact epithelial, since they expressed keratins. Also, S1 cells seemed to be a triploidation of a cell with close resemblance to S4, while only few cytogenetic differences were found between S4 and S8, suggesting an origin of S1 and S8 via S4 from a single hypothetical stem cell.     

Clonal chromosomal aberrations in simian virus 40-transfected human thyroid cells and in derived tumors developed after in vitro irradiation.

In vitro model cell systems are important tools for studying mechanisms of radiation-induced neoplastic transformation of human epithelial cells. In our study, the human thyroid epithelial cell line HTori-3 was analyzed cytogenetically following exposure to different doses of alpha- and gamma-irradiation and subsequent tumor formation in athymic nude mice. Combining results from G-banding, comparative genomic hybridization, and spectral karyotyping, chromosome abnormalities could be depicted in the parental line HTori-3 and in nine different HTori lines established from the developed tumors. A number of chromosomal aberrations were found to be characteristic for simian virus 40 immortalization and/or radiation-induced transformation of human thyroid epithelial cells. Common chromosomal changes in cell lines originating from different irradiation experiments were loss of 8q23 and 13cen-q21 as well as gain of 1q32-qter and 2q11.2-q14.1. By comparison of chromosomal aberrations in cell lines exhibiting a different tumorigenic behavior, cytogenetic markers important for the tumorigenic process were studied. It appeared that deletions on chromosomes 9q32-q34 and 7q21-q31 as well as an increased copy number of chromosome 20 were important for the tumorigenic phenotype. A comparative breakpoint analysis of the marker chromosomes found and those observed in radiation-induced childhood thyroid tumors from Belarus revealed a coincidence for a number of chromosome bands. Thus, the data support the usefulness of the established cell system as an in vitro model to study important steps during radiation-induced malignant transformation in human thyroid cells.