Trisomy of rat chromosome 1 associated with mesothelial cell transformation.

Identification of specific chromosomal aberrations in transformed mesothelial cells is an important step in elucidating the mechanism of transformation of these cells which are targets for occupational and environmental carcinogens, such as asbestos fibers. Cytogenetic analysis of normal rat mesothelial cell lines revealed that at late passage (p20-p34), trisomy of chromosome 1 was present in greater than 80% of the cells in four spontaneously immortalized lines examined, whereas at early passage (p8-p10), only 15-44% of the cells had trisomy 1. Trisomy of chromosome 1 had increased in the population as a function of passage, suggesting that cells with trisomy 1 had a selective growth advantage under in vitro culture conditions and that this alteration was associated with transformation. A commercially available rat mesothelial cell line (4/4 RM4, ATCC), was also found to have a duplication of a portion of the long arm of chromosome 1. To determine if chromosome 1 alterations have relevance to the transformed phenotype in vivo, a neoplastic cell line was established from a spontaneous rat mesothelioma. At passage 15, trisomy of chromosome 1 was observed in 26% of the metaphases in this line. However, when these cells were injected into nude mice, 99% of the cells from the resulting tumor contained an additional copy of chromosome 1. Therefore, trisomy 1 also conferred a selective growth advantage in vivo and/or was associated with the malignant subpopulation in the tumor derived cell line. These studies suggest that chromosome 1 contains a gene(s) involved in transformation of rat mesothelial cells.     

Random chromosome changes following SA7 transformation of Syrian hamster cells

The chromosome G banding technique was applied to Syrian hamster cells transformed in vitro by the carcinogenic virus SA7 or a combination of polycyclic carcinogenic hydrocarbon and SA7. Chromosome constitution of different clones varied; a normal diploid or an aneuploid constitution with minor structural or numerical deviation was found in some, while in others aneuploidy was associated with increased numerical changes as well as with abnormal chromosomes which are considered as markers. No consistent specific chromosome changes characterized transformation from SA7 or carcinogen pretreatment between the different pools of cells used. Although a lack of consistent chromosome pattern was associated with SA7 viral transformation, the possibility remains that extensive invisible minor genetic changes may have occurred.     

Cumulative gene and chromosome alterations associated with in-vitro neoplastic transformation of human cervical cells

The development of cancer is a multistep process requiring cumulative genetic alterations. An in vit ro model utilizing human cervical cells and papillomaviruses (HPV) that mimics human cervical cancer has been developed. Chromosome and gene alterations associated with distinctive stages of neoplastic transformation were demonstrated with an exocervical cell line obtained after sequential transfection with recombinant HPV-16 DNA and v-Ha-ras oncogene. Acquisition of immortality after HPV-16 transfection was associated with aneuploidy, structural changes of chromosomes 8, 10, 17, 19, 20, and 21, as well as proto-oncogene alterations. HPV-16 DNA was localized to two sites on chromosome 21, with one site at 21q22.2-22.3 near the ets-2 proto-oncogene. Ets-2 as well as c-myc gene mRNA levels were elevated in HPV immortal cells compared to primary nontransfected exocervical strains. Although the HPV-immortalized cells had several features characteristic of malignant cells, they lacked tumorigenic potential. Tumorigenicity occurred after transfection with v-Ha-ras oncogene, which was found stably integrated on chromosome 12 at the telomeric band q24.3. The tumorigenic line had additional clonal chromosomal abnormalities; consisting of multiple deletions involving regions of chromosomes 1p/q, 3p, 9q, loss of one copy of chromosome 11, and a complex rearrangement of chromosomes 8 and 13 as shown by in situ suppression hybridization with whole chromosome probes. Loss of tumor suppressor genes on deleted regions may have contributed to the acquisition of tumorigenicity. The genetic changes observed in these cells parallel those found in cervical carcinomas, demonstrating the validity of the in vitro model for studying the multistep progression resulting in cervical carcinoma.     

Tumor progression and transformation of low-grade glial tumors associated with pregnancy

Brain tumor growth or progression has been shown to occur in low-grade glial tumors and meningiomas. While progression has been documented in this population, transformation to a more aggressive high-grade glial tumor that can lead to increased morbidity and mortality has not been identified. In this case series, we document transformation from low-grade gliomas to high-grade gliomas (WHO grade III and IV) in young women during pregnancy. We further discuss the possible etiologies of this phenomenon.     

Trisomy of chromosome 9q: specific chromosome change associated with tumorigenicity during the process of X-ray-induced neoplastic transformation in golden hamster embryo cells

We have reported that trisomy of chromosome 7 is commonly observed in anchorage-independent clones isolated from X-irradiated golden hamster embryo cells. All 10 clones derived from different irradiated populations showed tumorigenicity when 1 x 10(7) cells were injected s.c. into nude mice (BALB/c, nu/nu). From karyotypic analysis, we found that 8 of 10 cells showed trisomy of chromosome 9. One cell line had a translocation between chromosomes 9q and 19q and trisomy of chromosome 7. The other cell line contained trisomy of chromosome 9 and a translocation between chromosomes 7q and 8q. Using Southern blot analysis, we observed no amplification of v-myc, v-Ha-ras, v-Ki-ras or N-ras-related oncogenes. Furthermore, we could not detect either an increase in expression of v-myc- and v-Ha-ras-related genes or the activation of any oncogene, by the NIH 3T3 transfection assay. Our results suggest that trisomy of chromosome 7 is insufficient for the expression of tumorigenicity and that increased dosage of chromosome 9q may play an important role in the malignant progression of X-ray-induced neoplastic transformation.     

Acute transformation of chronic large granular lymphocyte leukemia associated with additional chromosome abnormality

A patient with large granular lymphocyte (LGL) leukemia that transformed into an acute or aggressive form after 20 months of the chronic phase is reported. The patient's leukemic cells were mature, medium-sized lymphocytes with sparse azurophil granules and the surface phenotypes of the cells were CD2+, CD3-, CD11+, and CD16+. Molecular analysis showed a germ line configuration in both T-cell receptor beta-chain genes and T-cell receptor tau-chain genes. A clonal anomaly of chromosome (trisomy 8) was demonstrated in peripheral blood cells. LGL after acute transformation of the disease displayed large blastic morphology with prominent nucleoli, intense basophilic cytoplasm, and numerous granules. Karyotypic analysis demonstrated a mosaic of trisomy 8 and trisomy 8 with an additional marker chromosome. Thus, transformation of chronic LGL leukemia into an acute or aggressive form in this patient was associated with morphologic and karyotypic changes of the leukemic cells. Patients with a stable form of chronic LGL leukemia should be examined carefully for the possible acute crisis associated with a clonal evolution.     

Enhancement of viral and DNA mediated transformation of cloned rat embryo fibroblast cells by 3-aminobenzamide

We have analyzed the effect of the poly(ADP-ribose) synthesis inhibitor 3 aminobenzamide (3AB) on de novo and methyl methanesulfonate (MMS) and gamma irradiation enhancement of viral transformation of a cloned rat embryo fibroblast cell line, CREF, by a cold-sensitive host-range mutant of type 5 adenovirus, H5hr1. Additionally, we have evaluated the effect of 3AB on the transformation of CREF cells following transfection with a gene conferring resistance to hygromycin (hygr) or the neomycin analogue G418 (neor) in combination with a cloned type 5 adenovirus E1A transforming gene (Ad5 E1A) or the Ha-ras (T24) oncogene. 3AB induced a dose- and time-dependent increase in the level of de novo MMS-enhanced and gamma irradiation-enhanced transformation of CREF cells by H5hr1, whereas it did not induce morphological transformation in uninfected control, MMS-pretreated, or gamma irradiation-pretreated CREF cells. Temporal kinetic studies indicated that 3AB was most effective in enhancing de novo and MMS-enhanced and gamma irradiation-enhanced viral transformation when applied early after viral and carcinogen plus viral infection and when present for extended time periods (4-5 wk). 3AB also increased the frequency of resistant colonies following transfection with several cloned genes, including hygr, neor, and protein kinase C (which also contained a neor gene), and the frequency of morphological transformation of CREF cells following cotransfection with a hygr gene and an Ad5 E1A or an activated Ha-ras (T24) gene. In contrast, 3AB exerted the opposite effect, i.e., an inhibitory effect, when applied to NIH 3T3 cells transfected with a hygr or neor gene, alone or in combination with a T24 gene. The ability of 3AB to enhance the frequency of transformation of CREF cells was not associated with a selective effect on the growth of H5hr1-transformed CREF cells in monolayer or agar culture. Similarly, 3AB did not alter the percentage of MMS- or gamma irradiated-pretreated H5hr1-infected cells retaining free Ad5 DNA or the random pattern or quantity of viral DNA integration in control or carcinogen-treated H5hr1-transformed cells. These results suggest that cellular processes regulated by the nuclear enzyme ADPRT, or additional processes modified by 3AB, may be important mediators of stable transformation induced by transfected DNA and both de novo and carcinogen-enhanced viral transformation of specific target cells.     

Gene expression profiling of mouse teratocarcinomas uncovers epigenetic changes associated with the transformation of mouse embryonic stem cells

The molecular mechanisms of the development of teratocarcinomas from stem cells are largely unknown. To determine which genes are associated with the transformation of these cells, we have performed oligonucleotide microarray analysis, using Affymetrix U74A GeneChips, on both cell cultures and tumors in nude mice. We identified 68 genes that significantly differed in expression between the ES cell culture and the teratocarcinoma cell line, SCC-PSA1, and 51 genes with statistically different expression patterns between the ES cell tumors and the teratocarcinomas (P < .00005). We found that there were 20 genes that had common expression patterns in both groups. We also examined the role of the transition from in vitro to in vivo by comparing ES cell culture to ES cell tumor, and teratocarcinoma cell line to teratocarcinomas. We identified 22 genes that were upregulated in the ES cell tumors and 42 that had a decreased expression in the tumor (P < .0001). In comparing SCC-PSA1 to its tumor, we identified 34 upregulated genes and 25 downregulated genes (P < .001). There were only 10 genes in common from these two lists. GenMapp search revealed that several pathways, especially the cell cycle pathway, are actively involved in the induction of teratocarcinomas. Our results indicate that many key development genes may play a key role in the transformation of ES cells into teratocarcinoma cells.     

In vitro transformation of rat esophageal epithelial cells with N-nitrosobenzylmethylamine

Using an explant/cell culture system, rat esophageal epithelialcells were transformed in vitro by exposure to N-nitroso-N-benzyl-N-methylamine(BMNA). Twelve esophageal explant cultures per group were exposedtwice (at days 1 and 7) to 0.0, 2.5, 5.0 or 10.0 µg BMNA/mlof medium. After incubation for 60–90 days, epithelialcells in primary cultures treated with all three concentrationsof BMNA could be subcultured and cell lines were developed.The number of primary cultures and the number of subsequentlydeveloped epithelial cell lines was carcinogen-dose-dependent.Cell lines could only be established from carcinogen treatedexplants. Electron microscopy revealed that the BMNA-treatedcell lines contained morphological markers of esophageal epithelialcells; i.e., numerous tonofilaments and junctional complexes,even after prolonged subculture. By immunofluorescence, thecells reacted positively with antibodies prepared to mouse skinprekeratins (K₁ and K₂). Two cell lines (from the 5 µgBMNA/ml group) were able to grow in soft agar and produce palpabletumors upon injection into syngeneic recipients. These tumorspossessed the histological features of squamous cell carcinomas.     

Ruffling membranes in cultured human and rat glial cells

We have previously described how mouse monoclonal antibody Mab S-11E10 specifically stained ruffling membranes and filopodia of cultured human glioma cells. We found that this antibody stains ruffling membranes, not only of human astrocytes and glioma cells, but also of rat astrocytes and rat C6 glioma cells. In addition, we newly prepared rabbit polyclonal antibody for the antigens, which were eluted by Mab S-11E10 affigel 10 (Bio Rad) affinity column chromatography from cultured rat C6 glioma cells. The polyclonal antibody detects 65KDa, 57KDa, 46KDa, 43KDa bands in Western blotting analysis and specifically stained ruffling membranes of human glioma cells on cell spreading. Using the polyclonal antibody, we studied the change of the distribution of the antigens during cell spreading and in fully spread cells. At the beginning of cell spreading, ruffling membranes were specifically stained, but 72 hours after plating the immunofluorescence was localized in membranes of supranuclear regions and small protuberances. Furthermore, we semi-quantitatively measured the contents of the antigens detected by the polyclonal antibody, using dot blotting immunoassay. The contents of the antigens rapidly increased at the beginning of cell spreading and thereafter gradually reduced. The change of the contents seemed to be time--and cell density--dependent and to relate to the extent of cell spreading. These results suggest that the antigens may play an important role in cell spreading of glial cells.     

Membrane lipid changes associated with malignant transformation and normal maturation of human lymphocytes

Human lymphocytes from normal blood, normal thymus, chronic lymphocytic leukaemia blood and lymphoblasts from continuous cell lines were studied as representative of normal, immature and transformed lymphocytes in an attempt to determine whether a relationship exists between the lipid composition of the plasma membrane and the maturity or malignant transformation of the lymphocyte.Normal mature lymphocytes from peripheral blood contain more phospholipid and cholesterol per cell than immature thymocytes or chronic lymphocytic leukaemia cells suggesting that the normal maturation of lymphocytes is associated with an increase in the content of these lipids. Analysis of plasma membrane preparations from the four populations of lymphocytes showed that, when assayed on a per cell basis, the cholesterol : phospholipid molar ratio of malignant and transformed lymphocyte membranes is substantially lower than that from normal lymphocytes. In the case of chronic lymphocytic leukaemia cells this deficiency of membrane cholesterol may be due to a decreased ability of the cells to synthesise sterols. The fatty acid composition of the major membrane phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) also differed between the four lymphocyte populations studied.These observations of membrane phospholipid and cholesterol composition are consistent with previous assessments of the fluidity of the lipid core of normal and leukaemic lymphocyte membranes, but do not correlate with measurements of membrane dynamics using fluorescent antibody or lectins. This discrepancy is discussed.     

Neoplastic transformation of rat embryo cells with herpes simplex virus.

Sprague Dawley rat embryo cells (REF) were transformed by inoculation with herpes simplex virus (HSV) and incubation at 42 degrees C for 8 days. The infected cultures were subsequently returned to 37 degrees C and two types of cell clone were isolated from foci of growing cells after 4 weeks. One of the clones consisted of epithelial-like cells and did not produce HSV (REF-Tep-NP). The second consisted of spindle-shaped cells and cultures of these cells persistently developed small areas of degeneration where production of infectious HSV (REF-Tsp-P) took place. An additional clone which did not produce any more HSV (REF-Tsp-NP) was isolated from REF-Tsp-P in the presence of HSV-antiserum. REF-Tsp-P and REF-Tsp-NP grew more rapidly than REF and also formed foci in soft agar. REF-Tep-NP had a growth rate between that of normal rat embryo cells and that of both REF-Tsp-NP and REF-Tsp-P and did not form foci in soft agar. REF-Tsp-NP cells, in contrast to REF-Tep-NP cells, were resistant to superinfection with HSV types 1 and 2. REF-Tsp-P and REF-Tsp-NP produced metastasizing sarcomas in rats. After inoculation of 10(3) REF-Tsp-NP cells into 1-day-old rats tumours developed rapidly. REF-Tep-NP cells did not induce tumours in rats. The parental REF cells produced no tumours, even when 10(8) cells were inoculated into the rats. Positive immunofluorescence was observed in all three transformed cells only with the hyperimmune rabbit sera but not with human anti-HSV reconvalescence immune sera.     

Phagocytic capacity of normal and malignant rat glial cells in culture

The phagocytic capacity of 4 continuous rat glioma cell lines (BT2C, BT4Cn, BT5c, and 9L) and normal BD IX fetal rat glial cells in culture has been studied. This was done by flow cytometric measurements of single cells from monolayer cultures having ingested fluorescent bacteria, zymosan particles, red blood cells, or fragments of normal glial cells. In addition, phagocytosis was studied in a three-dimensional culture system. The BT4Cn, BT5C, and 9L cell lines were tumorigenic and invasive both in vivo and in organ culture in vitro. In contrast, BT2C has shown variable tumorigenicity and does not seem to be invasive. The phagocytic capacity of the cell lines was compared to their destructive properties during invasion. Depending on the particle type, 30-40% of the normal glial cells were phagocytic. The fractions of phagocytic glioma cells were dependent on the particle type and the prey load. Of the invasive cell lines, BT5C showed high phagocytic activity both in monolayer and three-dimensional cultures. Two of the invasive cell lines (BT5C and 9L) had about the same fraction of phagocytic cells as normal glial cells. These 2 cell lines showed highly destructive growth during invasion. In contrast, the third invasive cell line (BT4Cn) had almost no phagocytic cells. The BT4Cn cells showed single-cell invasion with little destruction of target tissue. The noninvasive cell line (BT2C) showed low phagocytic activity, and almost no destruction was observed in the border zone between tumor cells and normal tissue. Phagocytosis seems to be an inherent property of both normal and malignant glial cells, although the fraction of phagocytic cells varies from one cell line to another. In organ culture high phagocytic capacity of invasive glioma cells seems to be related to destructive activity on the normal brain tissue during invasion.