Characterization of a human ovarian adenocarcinoma line, IGROV1, in tissue culture and in nude mice

A cell line, IGROV1, originating from an ovarian carcinoma of a 47-year-old woman was established in tissue culture and in nude mice. Maintained in monolayer cultures, IGROV1 cells exhibited a 20-h doubling time and highly tumorigenic properties. The s.c. injection of 2 X 10(6) cultured cells into nude mice gave rise to fast growing tumors, while the i.p. route induced a peritoneal carcinomatosis with ascites which killed the animals in 2 months. The epithelial morphology of IGROV1 cells was retained during in vitro and in vivo passages, as judged by both the light and the electron microscopes. Two cytogenetic markers characterize IGROV1 cells: a paracentric inversion of chromosome 3, and a translocation between chromosomes 2 and 5. The constitutional karyotype of the patient was normal. These characteristics make the IGROV1 cell line a suitable experimental model for the treatment of human ovarian carcinomas and for biological studies of human solid tumors.     

In vitro studies of the cell cycle of mouse myelomonocytic leukemia using a fluorescence activated cell sorter and autoradiography

Cell cycle and various phase time of mouse myelomonocytic leukemia cells were studied in vitro with Fluorescence Activated Cell Sorter (FACS 420, Becton-Dikinson FACS Systems, Sunnyvale, CA, USA) in testing cellular DNA content and with autoradiography. At the same time, doubling time was calculated by counting the number of cells cultured in vitro within 6 days following implantation. According to the cellular DNA content, the proportions of various cell phases in the whole cell population were as follows: G1, S and G2 plus M phases take up 30.5%, 47.9% and 21.6%. Basing on autoradiography and direct counting, the cell number, the cell cycle, duration of various cell phases and doubling time were as follows: LI = 98.0%, MI = 1.7 +/- 0.9%, TG1 = 4.9 hrs, TS = 8.0 hrs, TG2 = 2.8 hrs, Tm = 0.3 hr, TC = 16.0 hrs, TD = 17.0 hrs. FACS assay showed that the duration of various cell phases are quite similar to that calculated by autoradiography. Therefore a simple and rapid FACS method may be widely used in the studies of cell cycle instead of autoradiography if estimating computer program is available.     

Establishment and characterization of human signet ring cell gastric carcinoma cell lines with amplification of the c-myc oncogene.

Two unique human signet ring cell gastric carcinoma cell lines (designated HSC-39 and HSC-40A) were established in vitro from the ascites of a 54-year-old male patient. Both cell lines were biologically quite similar, grew in vitro in suspension with a population doubling time of 28-30 h, and had cytological features of mucinous epithelial tumor cells. They formed colonies in soft agar, with a cloning efficiency of 0.8-1.0%. Ultrastructurally, numerous granules were observed in the cytoplasm, suggesting secretory activity. The frequent presence of desmosome and the tight junction at the cell boundary certifies the epithelial origin of the lines. Immunocytochemistry and radioimmunoassay showed production of tumor marker antigens (carcinoembryonic antigen, CA 19-9, and sialyl-Lex-i) and gastrin in both lines. These lines were transplantable in athymic BALB/c nude mice. The histopathology of each line growing in athymic BALB/c nude mice was similar to that of the original tumor. The karyotype of the cells was highly aberrant with structural and numerical changes. The presence of numerous double minute chromosomes and loss of the 13 chromosome and Y-chromosome characterize these lines. In addition, the amplified c-myc oncogene (16-32-fold) was found in both cell lines and original ascitic tumor cells. Overexpression of the c-myc mRNA was noted. These cell lines may be a useful tool, providing both in vivo and in vitro systems for further studies of the biology and therapy of human signet ring cell (or Borrmann's type IV carcinoma) gastric carcinoma.     

Suggestive evidence for functionally distinct, tumor-suppressor genes on chromosomes 1 and 11 for a human fibrosarcoma cell line, HT1080

One approach for identifying chromosomes which carry putative tumor-suppressor genes is the introduction of specific chromosomes into the tumor cells of interest. We examined the ability of human chromosomes derived from normal fibroblasts to suppress or modulate tumorigenicity in nude mice and the in vitro properties of HT1080, a human fibrosarcoma cell line. We first isolated mouse A9 cells containing a single human chromosome (1, 2, 7, 11, or 12) integrated with pSV2neo plasmid DNA. Following fusion of microcells from these A9 cells with the HT1080 cells, clones that were resistant to G418 were isolated and karyotypically analysed. Three of 4 microcell-hybrids with an introduced chromosome 1 were non-tumorigenic (#1-7, -8 and -13), whereas the parental HT1080 cells were highly tumorigenic. The other microcell-hybrid clone (#1-1) formed tumors, the cells of which had lost one copy of chromosome 1. Two clones from the #1-1 cells were isolated; one contained an extra copy of chromosome 1, and the other one did not. The former was non-tumorigenic and the latter was tumorigenic. The introduction of chromosome 11 also suppressed the tumorigenicity of HT1080 cells, while the introduction of other chromosomes, i.e., 2, 7, or 12, had minimal or no effect on the tumorigenicity of these cells. Cells from tumors formed by microcell-hybrids with the introduction of chromosome 2, 7, or 12 still contained the introduced chromosome. Interestingly, only the microcell-hybrids with an introduced chromosome 1 had an alteration in cellular morphology and modulation of in vitro transformed properties, i.e., cell-growth and saturation density in a medium containing 10% calf serum and cell-growth in soft-agar. Thus, the results indicate the presence of putative tumor-suppressor genes for HT1080 cells on chromosomes 1 and 11, and further suggest that the genes on these chromosomes control different neoplastic phenotypes.     

Diamine oxidase activity in human melanoma cell lines with different tumorigenicity in nude mice.

The activity of diamine oxidase (DO, EC 1.4.3.6.) which converts putrescine into gamma-aminobutyraldehyde in the degradative pathway of polyamine, was studied in 4 human melanoma cell lines, 2 of which produce tumours in greater than 80% of nude mice (M3Dau, M4Beu), whereas the other 2 induce tumours in less than 25% (M1Dor, M2GeB). The activity of DO in these cells varies with the growth rate: 24 h after seeding there is an initial increase in DO activity, followed by a steep decline during exponential growth. At 96 h, when cells reach saturation density, the activity of DO is significantly greater in the highly tumorigenic cell lines than in the poorly tumorigenic cell lines. Kinetic studies show that for the highly tumorigenic lines apparent Km values are 10.6 X 10(-6)M +/- 0.2 (M3Dau) and 14.2 X 10(-6) M +/- 0.6 (M4Beu), whereas for the poorly tumorigenic lines the values are 4.5 X 10(-6) M +/- 0.3. After transplantation into nude mice, the M1Dor cell line, which exhibits a low Km (app.) for DO of which had high Km (app.) value. Km (app.) determination of DO could be an approach for characterizing human melanoma cells differing in their tumorigenic potential in nude mice.     

Suppression of tumorigenicity of rat liver tumor cells by human chromosome 13: evidence against the involvement of pRb and BRCA2.

Aberrations of chromosome 13, including large-scale deletions and rearrangements, have been implicated in the development of a significant fraction of human hepatocellular carcinomas, suggesting that liver tumor suppressor genes may be located on this chromosome. In this study, we have employed a microcell hybrid-based model system to investigate the presence of liver tumor suppressor loci on human chromosome 13. The parental GN6TF rat liver epithelial tumor cells are highly tumorigenic in vivo and exhibit altered cellular morphology and growth characteristics in vitro. The GN6TF cells form tumors in 100% of syngeneic animals with short latency, are not contact inhibited or anchorage-dependent in cell culture, and do not express mRNAs for rat Rb1 and BRCA2. Microcell-mediated introduction of human chromosome 13 into the rat liver tumor cell line GN6TF resulted in the generation of clonal microcell hybrid (MCH) cell lines that differentially exhibited tumor suppression and/or alteration of other transformation-associated phenotypes in vitro. Two GN6TF-13neo MCH lines exhibited characteristics indicative of suppression by the human chromosome, including a normalized cellular morphology and growth pattern, loss of anchorage-independent growth potential, partial restoration of contact inhibition, reduction in tumorigenic potential in vivo, and dramatic elongation of tumor latency. In contrast, three GN6TF-13neo MCH cell lines were minimally affected by the introduction of the human chromosome and were nearly indistinguishable from the parental GN6TF tumor cells, exhibiting a highly aggressive tumorigenic phenotype in vivo. Both suppressed and non-suppressed GN6TF-13neo MCH cell lines express Rb1 and BRCA2 mRNA in vitro, and tumors derived from the non-suppressed GN6TF-13neo MCH cell lines continue to express Rb1 and BRCA2 mRNA in vitro, and express pRb in vivo. The results suggest that: i) human chromosome 13 contains a liver tumor suppressor locus, ii) expression of Rb1 and/or BRCA2 is insufficient to produce tumor suppression in this rat liver tumor cell line, and iii) that the human chromosome 13 liver tumor suppressor may represent a novel tumor suppressor gene, distinct from Rb1 and BRCA2.     

Tumorigenic transformation of spontaneously immortalized fibroblasts from patients with a familial cancer syndrome

Immortal cell lines arose spontaneously during in vitro culture of initially normal fibroblasts, MDAH041 and MDAH087, from patients with Li-Fraumeni familial cancer syndrome. Fibroblasts from a control donor, MDAH170, maintained a normal morphology and senesced at 31 population doublings. The immortal fibroblasts have several properties of transformed cells. In addition to having acquired an altered morphology and chromosomal anomalies, MDAH041 and MDAH087 have escaped from senescence, growing beyond 300 and 100 population doublings (pd), respectively. As early as 50 pd, these cells can be transformed by an activated H-ras oncogene to form tumors in nude mice. However, MDAH041 immortal cells were resistant to tumorigenic transformation by transfection with the v-abl oncogene.     

Syngeneic immune response to rat tracheal epithelial cells transformed in vitro by N-methyl-N-nitro-N-nitrosoguanidine.

Two cell lines (2-10-1 and 8-10-2) derived by exposure to primary tracheal explants to MNNG in vitro were not tumorigenic in syngeneic F-344 rats or athymic BALB/c (nu/nu) mice at early passage, but became tumorigenic at late passage. These cell lines are therefore suited to study the expression of neoantigens during neoplastic development. Transplantation resistance to late-passage, tumorigenic cells was indicated in syngeneic rats using an immunization protocol of repeated cell inoculation and tumour ablation. Spleen cells from such animals were reactive in 20h microcytotoxicity assays against neoplastic cell lines, but unreactive to normal tracheal epithelial cells. Similarly, immune spleen cells co-cultivated in vitro for 6 days with irradiated neoplastic cell lines before assay for microcytotoxicity were strongly reactive, whereas co-cultivation with normal epithelial cells did not stimulate reactivity. Antibody to these neoplastic cell lines was demonstrated in sera of tumour-resistant rats by an indirect radiolabelled-antibody binding test and by indirect immunofluorescence. There was no significant binding to normal tracheal epithelial cell outgrowths.     

Characterization of variant subclasses of cell lines derived from small cell lung cancer having distinctive biochemical, morphological, and growth properties

We have described the establishment and biochemical characterization of 50 small cell lung carcinoma (SCLC) cell lines. Further analysis of these data, combined with studies of morphology and growth characteristics, indicates that 35 (70%) of the lines retained typical morphology (SCLC, intermediate subtype), growth characteristics (growth as tightly packed floating cellular aggregates, long doubling times and low colony-forming efficiencies), and biochemical profile (presence of L-dopa decarboxylase, bombesin-like immunoreactivity, neuron-specific enolase, and high concentrations of brain isoenzyme of creatine kinase). They are referred to as classic SCLC lines. The remaining 15 (30%) lines had discordant expression of the biochemical markers; they retained high concentrations of brain isozyme of creatine kinase, but had significantly lower concentrations of neuron-specific enolase and lacked L-dopa decarboxylase and bombesin-like immunoreactivity. These cell lines are called variants. SCLC variant lines could further be divided into (a) biochemical variant lines having variant biochemical profile but retaining typical SCLC morphology and growth characteristics; and (b) morphological variant (SCLC-MV) lines having variant biochemical profile, altered morphology (features of large cell undifferentiated carcinoma) and altered growth characteristics (growth as loosely attached floating aggregates, relatively short doubling times and cloning efficiencies). Fifty-five clones derived from the three SCLC subclasses retained their parental phenotypes. In SCLC-MV lines there was a near constant relationship between variant morphology, altered growth characteristics and amplification of the c-myc oncogene; classic SCLC and biochemical variant SCLC lines were not amplified. Variant morphologies frequently are present in SCLC tumors at autopsy, and most SCLC-MV lines reflect changes that had occurred in the tumors from which they were derived. Because SCLC-MV tumors behave more virulently in the patient and are radioresistant in vitro, these findings are of considerable biological and clinical interest.     

Establishment and characterization of TSGH9201, a human gastric carcinoma cell line that is growth inhibited by epidermal growth factor

A human signet ring gastric carcinoma cell line TSGH9201 was established in vitro. The cells grew in vitro as a monolayer with polygonal morphology and had a population doubling time of 34 hours. The cells secreted tumor markers CEA and CA 125. They were, however, not tumorigenic in athymic nude mice. Karyotypic analysis demonstrated a near tetraploidy with a modal chromosome number of 98. Northern blotting and immunocytochemical analysis revealed the expression of both transforming growth factor α and high levels of epidermal growth factor receptor. Cell growth was inhibited by the epidermal growth factor in vitro. The cell line may be a useful tool to study autocrine growth regulation through the epidermal growth factor receptor. © 1995 Wiley-Liss, Inc.     

Telomere shortening, telomerase expression, and chromosome instability in rat hepatic epithelial stem-like cells

Telomeres, which are specialized structures consisting of T2AG3 repeats and proteins at the ends of chromosomes, may be essential for genomic stability. To test whether telomere length maintenance preserves genomic stability in rats (Rattus rattus and Fischer 344), we assayed telomerase activity and telomere length in the rat hepatic epithelial stem-like cell line WB-F344 during aging in vitro and in tumor-derived lines. Telomerase activity in the parental WB-F344 line was repressed at low and intermediate passage levels in vitro and reexpressed at high passages. Southern blot hybridization and quantitative fluorescence in situ hybridization analyses demonstrated that telomeres were significantly eroded at intermediate passage levels, when telomerase was repressed, and at high passage levels, when telomerase was expressed. Fluorescence in situ hybridization analysis also revealed interstitial telomeric sequences in rat chromosomes. Tumor-derived WB-F344 cell lines that express telomerase had variably shortened telomeres. Cytogenetic analyses performed on WB-F344 cells at low, intermediate, and high passages demonstrated that chromosome instability was most severe in the intermediate passage cells. These data suggest that telomere shortening during aging of rat hepatic epithelial stem-like WB-F344 cells in vitro and during selection of tumorigenic lines in vivo may destabilize chromosomes. Expression of telomerase in high passage cells appeared to partially stabilize chromosomes.     

ETN-1: a new human endometrial carcinoma cell line producing ascites and distant metastases in nude mice

A human carcinoma cell line (ETN-1) has been established from a skin metastasis of a moderately differentiated adenocarcinoma of endometrial origin. The cell line has been so far maintained for 27 months through 55 passages, growing as a monolayer as well as in 3-dimensional clusters with a population doubling time of 72 hr. The number of chromosomes per cell varied from 39 to 107 (average number 61.0 +/- 19.8), with a modal number of 46-48. Seven clonal marker chromosomes were detected. Flow cytometric analysis revealed a population of pseudo-tetraploid cells (DNA index 2.1) next to a pseudo-diploid population (DNA index 1.1). The epithelial character of the cells was confirmed by a positive immunocytochemical reaction using monoclonal antibodies (MAbs) to different keratins, the epithelial cell markers BW 495/36 and HMFG-2, as well as by the presence of many junctional complexes. The tumour cells retained a positive reaction with the anti-ovarian carcinoma OV-TL 3, OV-TL 10 and OC 125 MAbs, although the reaction was markedly diminished in comparison with the original tumour. Tumour cells inoculated subcutaneously in nude mice produced well differentiated adenomatous tumour nodules with formation of glandular lumina and basal lamina. Tumour cells injected intraperitoneally produced malignant ascites and regional as well as distant metastases of adenomatous character.     

Establishment, characterization and virus expression of cell lines derived from radiation- and virus-induced lymphomas of C57BL/Ka mice.

Permanent cell lines have been established in vitro from lymphoid tumors induced in C57BH/Ka mice by fractionated X-irradiation or by inoculation of the radiation leukemia virus (RadOV). The cultured cells are lymphoblastic, replicate rapidly in vitro, and are tumorigenic in vivo. The cell surface markers Thy 1, Ly 1, Ly 2,3 and GIX are expressed by the lymphoid tumor cells in the mouse and persist in the corresponding cell lines; expression of the H-2 and TL antigens is greatly reduced during in vitro passage, but is restored on in vivo transplantation. The cell lines derived from RadLV-induced tumors (BL/VL lines) produce a virus population (RadLV/LTC) with the thymotropic and leukemogenic attributes of RadLV. Those derived from radiation-induced, virus-negative lymphomas (BL/RL lines) are initially devoid of MuLV expression, but frequently become spontaneous virus producers during in vitro cultivation.     

Establishment and characterization of two new cell lines derived from squamous cell carcinoma of the tongue in Chinese patients.

Two new cell lines derived from squamous cell carcinoma of the tongue, T1/CUHK and T2/CUHK, have been established in culture. Analysis of the morphology, ultrastructure, chromosome number, spheroid formation and immunohistochemical properties of the two cell lines demonstrated that they are both well characterized. T1/CUHK cells grew relatively faster than T2/CUHK cells. Both cell lines were tumorigenic after inoculation into made mice and showed positive reactivity with HPV 16 DNA probe. The reactivity of both cell lines with HPV 18 DNA probe was weak.     

Isogenic human mammary epithelial cell lines: novel tools for target identification and validation

Tumorigenesis is a multi-step process involving several consecutive genetic alterations resulting in loss of genomic stability and deregulated signal transduction pathways. To study these deregulated processes in vitro, typically established cancer cell lines derived from primary tumors, ascites, or from metastases are used. However, these cancer cell lines reflect only late stages of the tumorigenic process. To better understand the consequences of the sequential genetic alterations in an in vitro model system, we applied consecutive immortalization and transformation of primary human mammary epithelial cells (HMECs) combining shRNA-mediated knockdown of tumor suppressor genes and overexpression of oncogenes. Thereby, we developed a panel of isogenic HMEC-derived cell lines reflecting the multi-step process of tumorigenesis. The immortalized cell lines have a normal epithelial morphology and proliferate indefinitely and anchorage-dependently. In contrast, the transformed cells exhibit mesenchymal-like morphological changes and strong colony-forming activity in soft agar. SNP array analysis showed that none of the cell lines displayed gross chromosomal aberrations in 80 % of the chromosomes. However, massive changes were observed in some chromosomes of the transformed cells indicating that the transformed phenotype is characterized by chromosomal alterations. The isogenic immortalized and transformed cells described here provide a powerful tool for the in vitro validation of target genes for cancer therapy.     

Establishment and characterization of continuous cell line MTC-SK derived from a human medullary thyroid carcinoma

Tumor cells of a human medullary thyroid carcinoma were isolated and propagated in tissue culture. Several cell lines with different morphology developed from the primary culture, among others a fibroblast-like growing cell line (MTC-F) and a cell line growing as a suspension of single cells and spherical cell clusters (MTC-SK). The MTC-SK cell line was serially propagated for 90 passages, over 3 years. When examined at different times throughout the in vitro period, MTC-SK exhibited properties characteristic of medullary thyroid carcinomas: the cells maintained their epithelioid morphology; endocrine granules were demonstrated in the cytoplasm by electron microscopy; in situ hybridization confirmed the production of calcitonin- and bombesin-mRNA (gastrin releasing peptide); the cells revealed positive immunoreactivity with antibodies to calcitonin, calcitonin gene-related peptide, and bombesin. The in vitro properties of the MTC-SK cells corresponded to the results obtained from the tissue of origin. Cytogenetic studies of the MTC-F cell line revealed a supernumerary metacentric chromosome (20?). In the MTC-SK cell line the predominant findings were terminal chromosomal rearrangements most frequently concerning chromosome 11p, i.e., the locus of the calcitonin and calcitonin gene-related peptide genes and the H-ras oncogene, and a characteristic instability of the centromeric region of chromosome 16 and somatic pairing of the homologous chromosomes 16.     

JAK kinases overexpression promotes in vitro cell transformation

Constitutive activation of the JAK-STAT pathway is frequent in cancer and contributes to oncogenesis. Here, we took advantage of the Ba/F3 cell line, a murine proB cell line dependent on IL-3 for growth, to analyse mechanisms of constitutive STAT activation in vitro. Cytokine-independent and tumorigenic Ba/F3 cell lines were derived from a two-step selection process. Cells transfected with a defective IL-9 receptor acquire IL-9 responsiveness during a first step of selection, and progress after a second selection step to autonomously growing tumorigenic cells. Microarray analysis pointed to JAK1 overexpression as a key genetic event in this transformation. Overexpression of JAK1 not only increased the sensitivity to IL-9 but also allowed a second selection step toward cytokine-independent growth with constitutive STAT activation. This progression was dependent on a functional FERM and kinase JAK1 domain. Similar results were observed after JAK2, JAK3 and TYK2 overexpression. All autonomous cell lines showed an activation of STAT5, ERK1-2 and AKT but only TYK2-overexpressing cell lines showed a constitutive activation of STAT3. Thus, JAK overexpression can be considered as one of the oncogenic events leading to the constitutive activation of the JAK-STAT pathway.     

Specific cytogenetic abnormalities in two new human colorectal adenoma-derived epithelial cell lines

Two new epithelial cell lines from sporadic human colorectal adenomas designated S/AN and S/RG are reported. S/AN was from a villous adenoma and S/RG from a tubular adenoma. Both cell lines have extended growth capacities in vitro reaching passages 18 and 15, respectively, so far and show no signs of senescence. S/AN and S/RG have retained in vitro the ability to form mucin-producing goblet-like cells. Every cell of S/AN has a deletion on the short arm of chromosome 1 and one normal copy of chromosome 1. S/AN is also monosomic for chromosome 18. The majority of cells of S/RG only have one normal copy of chromosomes 6, 7, 14, 17, 18, and 22. S/RG also has several marker chromosomes. Although aneuploid S/AN and S/RG are nontumorigenic in athymic nude mice, these cytogenetic abnormalities are insufficient for the fully tumorigenic phenotype. The common abnormality for S/AN and S/RG is monosomy for chromosome 18, indicating that this is a central and important step in colorectal carcinogenesis. Our cytogenetic analysis of the adenoma cell lines suggests at least two possible routes by which premalignant colonic cells can develop and progress to malignancy. S/RG, unlike most other adenoma cell lines, is clonogenic. Aneuploidy, clonogenicity, and extended in vitro growth capacity may therefore be useful in vitro markers for adenoma cell lines with a relatively high malignant potential.