Heteroploid Cell Lines Research Articles

In vitro Activity of the Novel Pyrimidines and Their Condensed Derivatives Against Poliovirus

Background: Substituted pyrimidine derivatives (non-nucleoside) are found to be associated with various biological activities. The various substituted pyrimidines are also having significant in vitro activity against different DNA and RNA viruses. The present study focuses on the anti-PV activity of new pyrimidines and their condensed derivatives. Methods: A series of novel pyrimidines and their condensed derivatives were synthesized and their structures were confirmed by spectral data. Their antiviral activities against poliovirus type 3 (PV-3) were evaluated in vitro. In cell culture, morphological changes observed in cells infected with polioviruses, including cell rounding and detachment from the substrate, are generally termed cytopathic effects (CPE). The effects of synthetic pyrimidines on PV amplification in a culture of the heteroploid cell line, Vero 76 (African green monkey kidney cells) were investigated. Results: Bioassays in vitro showed that one of seven synthesized compounds, 7-(Benzenesulfonyl)-5- benzyl-N-(prop-2-en-1-yl)-5H-pyrrolo[3,2-d]pyrimidin-4-amine, has potent antiviral activity against PV-3 (EC50 = 0.75 μM). The selectivity index of this compound is similar to that of pirodavir. Conclusion: The need for antiviral agents to treat PV-associated diseases remains great, but few options currently exist. Here we show that substituted pyrimidine derivatives are a promising structure class of chemical compounds for the development of antiviral drugs against PV infections.

More on the Lack of Correlation between Terra Expression and Telomere Length

More on the Lack of Correlation between Terra Expression and Telomere Length

Silver staining and nucleolar patterns in human heteroploid and measles-carrier cells.

Silver staining revealed 10, 11 and 7 active rDNA sites (Ag-NORs) in acrocentrics of human diploid fibroblasts, near-triploid Lu106 cells (a HeLa-derivative) and measles-carrier Lu106 cells, respectively. Thus, the heteroploid cell lines showed no significant increase in Ag-NORs. They also had consistent Ag-staining patterns, in accordance with their karyotypic uniformity. The numbers of Ag-NORs agreed with the numbers of acrocentrics present. The fewer Ag-NORs in the measles-carrier cells showed increase of the Ag-stained material indicating regulated ribosomal gene activity. Similarly, in interphase cells stained with toluidine blue, the nucleoli were fewer but larger in the measles-carrier cells. By combining Ag-staining and G-banding in control and measles-carrier Lu106 cells, it was possible to identify the Ag-positive acrocentrics. These included normal and marker chromosomes. In both cell lines. Ag-positive chromosomes No. 15 were frequent. Numbers and sizes of nucleoli at anaphase, telophase and early interphase in monolayers corresponded to the Ag-staining patterns in air-dried metaphases. Thus, silver-stained regions in the metaphase chromosomes develop and grow into larger nucleoli during interphase. Ag-staining of some interphase nuclei in the measles-carrier cells comprised materials other than those found at the primary NORs.

Stability of the prion protein-encoding (PRNP) gene in HeLa cells

To assess the risk of the de novo emergence of the agent of transmissible spongiform encephalopathies in cultured cells, we examined the stability of the prion protein-encoding (PRNP) gene in HeLa cells and in cultures contaminated with HeLa cells that have been passaged extensively for over 50 years. Various sub-lineages of HeLa cells showed that some contained a mixture of a truncated PRNP gene (R3–R4 deletion) and a full-length PRNP gene, while others were homozygous for the R3–R4 deletion. That finding suggests that the progenitor of several popular sub-lineages of HeLa must have lost part or all of chromosome 20 early in the history of HeLa cells. No mutations were found in the PRNP genes. We conclude that the spontaneous appearance of mutations leading to expression of abnormal prion proteins in continuously passaged heteroploid cell lines is unlikely to pose a substantial risk for the safe production of biologicals in such cells.

Detecção da citotoxicidade de materiais biocompatíveis nas linhagens celulares MRC-5, HeLa e RC-IAL

The sensitivity of diploid and heteroploid cell lines for detection of cytotoxicity using the agar diffusion method on cell culture, was tested with ascorbic acid solution of different concentrations. A total of 562 samples of 21 various materials were tested. The heteroploid cell line, RC-IAL, showed in relation to the MRC-5 and HeLa cell lines, greater sensitivity because it showed the presence of cytotoxic effect with the lowest concentration used (10 and 25 micrograms/ml) of ascorbic acid and showed greater diameter of cytotoxic halo in 15 samples and equal diameter in 16 of the 43 positive samples (7.6%). Out of 43 positive samples, the MRC-5 line did not show cytotoxicity in 3 sponge samples and 1 of acrylic resin. The PVC (polyvinylchloride) and polyethylene rarely showed positivity, while, the plastic, cotton and acrylic resin demonstrated cytotoxicity in about 5% of samples. We thus suggest the use of the RC-IAL and HeLa cell lines for continuation of this type of analysis at Adolfo Lutz Institute.

Non-random distribution of abnormal mitoses in heteroploid cell lines.

We have performed absorption-cytometric DNA measurements of the DNA contents of the G0/G1, G2, metaphase, and telophase cells of the heteroploid MCa-11 and HL-60 lines, as well as the WCHE-5 line which has a narrowly restricted number of chromosomes. We found that morphologically unbalanced mitoses occurred much more frequently in telophase-cell pairs of the heteroploid MCa-11 and HL-60 lines than in those of the chromosomally stable WCHE-5 line. Furthermore, the morphologically unbalanced mitoses represented unequal segregation of DNA into each of the daughter telophase nuclei. Such mitotic segregation errors (MSE) occurred almost exclusively in telophase cells with DNA contents which were above, or below, the DNA content of the modal telophase population. The net effect of these non-random, unblanced divisions of heteroploid cells with non-modal DNA contents is to produce one daughter cell with a DNA content that tends to return to the modal DNA content peak.

Discrepancies among the metaphase, telophase, and the G0/G1 and G2 DNA peaks of heteroploid cell lines

Heteroploid cell populations often show narrow peaks of G0/G1 and G2/M DNA content and broadly distributed chromosome numbers. This was originally explained by the selective metaphase arrest of the cells that have non-modal chromosome numbers. To test whether this explanation applies, we have measured the chromosome number distributions, as well as the G0/G1, G2, metaphase (M), and telophase (T) DNA distributions, of the cell lines WCHE-5, MCa-11, and HL-60. The WCHE-5 cells had narrowly distributed chromosome numbers and G0/G1 G2, M, and T DNA peaks. The MCa-11 and HL-60 cells also had narrowly distributed G0/G1 and G2 DNA peaks, but broadly distributed chromosome numbers and M and T DNA peaks. The widths of the MCa-11 and HL-60 M- and T-cell DNA peaks were similar to those of their chromosome number peaks, suggesting that all cells were completing mitosis, regardless of chromosome number or DNA content. Thus, selective metaphase arrest does not seem to be the cause of the narrow G0/G1 and G2 DNA peaks of heteroploid cell populations.

A heteroploid permanent cell line originating from embryonic calf thyroid supporting the replication of all known bovine adenovirus serotypes

An embryonic calf thyroid cell culture was established as a permanent heteroploid cell line, which is now in its 150th subculture. It allowed replication of all nine bovine adenovirus serotypes at its 15th as well as its 60–150th passages. All viruses induced typical cytopathic effects. Yields obtained on the permanent calf thyroid line were, on average, 0.8 log 10 lower than those obtained on primary calf testicle cells.

WiDr is a derivative of another colon adenocarcinoma cell line, HT-29

This paper documents chromosomal and isozymic evidence indicating that WiDr is a derivative of another colon adenocarcinoma cell line, HT-29, which was established in 1964. The phenotypes for seven isozymes studied were identical for all HT-29 and WiDr cell lines. The karyotypes were similar between four HT-29 cultures separated by more than 100 passages and two WiDr cultures that were studied independently in 1979 and 1985. The isozymic and karyotypic data are consistent with that described for the cell line WiDr, originally reported in 1979. Detailed karyotypic comparisons further show that cultures of WiDr possess all nine chromosomal markers found in the initial HT-29 (passage 19) cultures. However, both the higher passaged HT-29 and WiDr differ from the initial HT-29 (passage 19) because it has an additional five to eight constitutive marker chromosomes. An increase or decrease of one copy in several normal chromosomes is also noticed with more normal chromosome gains than losses. The study provides another example demonstrating the remarkable karyotypic stability within a heteroploid cell line with continued passage. It also reemphasizes the critical need for detailed karyotypic and isoenzymic studies on all cell lines in common use.

DNA Content of Diffusely Infiltrative Carcinomas in the Stomach

DNA content of diffusely infiltrative carcinomas of 15 Japanese patients, ranging in age from 38 to 65 years, was determined by cytofluorometry, using paraffin sections which were available for histological examination. Sections were stained with 0.0025% propidium iodide. By measuring the fluorescence intensity of at least 50 mitotic cells, ploidy patterns of the cancers were determined, whereas a control of the diploid DNA content was set by measuring the fluorescence intensity of mitotic cells in the non-cancerous gastric mucosa; only metaphase nuclei in the sections were considered to have a whole amount of nuclear DNA in the cytofluorometric measurement. In the present study, it was found that most of the diffusely infiltrative carcinomas consisted of a heteroploid cell line. Their stem DNA contents ranged between 2.8C and 4.8C (2C corresponds to a diploid amount of nuclear DNA). Of 15 cancers, four comprised a mosaic cancer of two different heteroploid cell lines. We encountered only one carcinoma containing a diploid cell line. However, this cancer also contained a heteroploid cell line. Six cancers contained a polyploid cell population, the DNA content of which was double of the stem DNA content. In most cases, the cancer cells distributed in the mucosal layer were also heteroploid, thereby suggesting a heteroploid origin of the diffusely infiltrative carcinomas.

Clones of MRC-C cells may be superior to the parent line for the culture of 229E-like strains of human respiratory coronavirus

A clone was selected from the MRC-C continuous heteroploid cell line which was significantly better than MRC-C for the culture of human respiratory coronavirus (HCV) 229E, strain LP. Such clones could prove generally useful for the isolation of HCV-229E from clinical specimens, and for the propagation and assay of laboratory adapted strains.

Freeze-dried microsomes: induced metabolic activation of cyclophosphamide as a cause of cytogenic damage to a human heteroploid cell line

Freeze-dried microsomes: induced metabolic activation of cyclophosphamide as a cause of cytogenic damage to a human heteroploid cell line

Silver staining in Drosophila melanogaster: NOR behaviour in heteroploid cultured cells.

A heteroploid cell line ofDrosophila melanogaster was cytologically analyzed by silver staining to investigate the in vitro behaviour of NORs. A single Ag-positive NOR was detected in all the metaphases observed, suggesting a possible suppression of excess ribosomal genes.

Frequencies of Sister Chromatid Exchanges in Heteroploid Cell Lines of Human Melanoma Origin<xref ref-type="fn" rid="fn2">2</xref>

Sister chromatid exchange(s) (SCE) per cell and chromosome in five heteroploid cell lines and sublines of human melanoma origin were distinctly more numerous than those in the normal diploid control. The SCE per unit chromosome length was higher in stable heteroploid C32 (0.041-0.047) and C9 (0.056) than in normal diploid control. The SCE per unit chromosome length was higher in stable heteroploid C32 (0.041-0.047) and C9 (0.056) than in normal diploid control C182 (0.036), which suggested that the longer the total chromosome length of the cell genome, the higher the SCE per cell as well as per unit chromosome length. These malignant heteroploid lines had karyotypes ranging from hypodiploidy to hypotriploidy and were extremely heterogeneous in one cell line but unusually homogenous and stable in others. However, none of these properties could be correlated to the increase in SCE frequency. As compared to C32-r7, the subline C32-RO had more SCE per cell, chromosome, and unit chromosome length for the genome as well as for 9 of 11 individual chromosomes and chromosome groups. In contrast, the C32-RO specific ring marker chromosome had only 40% of the SCE of its nonring homologue in C32-r7 cells. Interestingly, C32-RO and its ring marker were less stable than C32-r7 and the nonring marker during in vitro growth. There was no noticeable difference between C32-r7 and its derivative C32-r7-nu-1, which implied that transplantation into nude mice did not alter the expression of SCE in this subline.

Replication of human syncytium-forming virus in human cells: effect of certain biological factors and selective chemicals.

The growth characteristics of the human syncytium-forming virus (HSFV) were examined in several human cell lines of normal and malignant origins and composing of either fibroblastic or epithelial-like cells. Virus production occurred only in the fibroblastic diploid cell lines: HEF (human embryonic cells, Flow #5,000) and HFDL #645 (human fetal diploid lung), but not in the epithelial-like heteroploid cell lines: RA (a continuous line of human amnion), #999 (human bone marrow), and KB (carcinoma of the nasopharynx). While the single-cycle growth pattern of the virus in HEF and HFDL #645 cell lines were essentially similar, the virus yield per cell was greater in the HFDL #645 cells. Furthermore, the physiological state of the cell had a marked effect on virus production. Subconfluent actively growing HFDL #645 cells produced higher yields of virus than density-inhibited confluent HFDL #645 cell cultures. The replication of HSFV was inhibited by actinomycin D at concentrations that did not interfere with poliovirus replication (0.001 to 0.01 microgram/ml). Pretreatment and posttreatments of infected cell cultures with either the polycation polybrene (hexadimethrine bromide) or the synthetic glucocorticosteroid dexamethasone did not enhance HSFV production.

Quantitation of cell DNA in the evaluation of heteroploid cells as substrate for the preparation of killed viral vaccines.

To evaluate the risk of using heteroploid cell lines as substrates for viral vaccine production, the presence of cell DNA in poliovirus suspensions was examined. The time course of [3H]-thymidine-labeled HeLa cell DNA release during lytic infection with type 1 poliovirus was investigated. More DNA was found in filtered supernatants of poliovirus-inoculated cultures than in control cell supernatants. DNA concentration increased with time, paralleling virus release, but did not exceed 1.5% of the total DNA content of the culture. Only about 10% of this cell DNA was resistant to DNase treatment. By both ion-exchange chromatography and rate-zonal centrifugation it was possible to remove practically all cell DNA contaminating filtered poliovirus suspensions. Results obtained in this study permitted quantitative evaluation of cell substrate DNA present in poliovirus suspensions during successive steps of killed poliovirus vaccine preparation. Based on the sensitivity of our method, the amount of residual DNA was estimated at less than 0.02 pg per dose of purified vaccine.

Cytotoxic and cytogenetic effect of nitrogen mustard on EUE cells pretreated with sodium warfarin

A combined treatment with warfarin (W) and the alkylating agent nitrogen mustard (HN 2) was performed from the human heteroploid cell line (EUE). Warfarin was added to the cultures and after 24 hr contact the W treatment was repeated together with different HN 2 doses. Cytotoxicity was measured as percentage reduction in cell density and the chromosomal effects were evaluated by determining the frequency of chromosomal aberrations. As compared to EUE cultures treated only with HN 2 a significant increase in the toxicity values and in the amount of cytogenetic damage was found in cultures submitted to a combined treatment of HN 2 and W, which does not display toxicity in the experimental conditions utilized.

DNA content and chromosome number of a heteroploid human tumour cell line

In this paper investigations concerning the relation between variability of chromosome number and variability of DNA content within the cells of a tumour stemline are reported. A highly heteroploid human tumour cell line was used, which was derived from a chondrosarcoma. Flow cytometrical and scanning cytophotometrical measurements confirmed the heteroploid nature of the original cell line and of several subclones. Measurement of the DNA content per metaphase showed a linear relation between chromosome number and DNA content of heteroploid cells. This finding is discussed with regard to its implications for the mechanism of heteroploidy in tumour cells.

Difference between diploid and aneuploid Chinese hamster cells in replication at mid-S-phase.

Following partial synchronization of the heteroploid Chinese hamster cell line V-79 and of normal diploid lung fibroblasts of the Chinese hamster in culture, their DNA replication during S-phase aws compared by means of a BrdU-incorporation/thymidine pulse technique and Hoechst-Giemsa differential staining of metaphase chromosomes. This comparison indirectly shows the S-phase of the heteroploid cells of V-79 to be 2 h shorter than the diploid cell S-phase. When the thymidine pulse is applied to diploid lung fibroblasts at mid-S-phase, differential staining colours metaphase chromosomes a pale blue. Performing the corresponding experiment with V-79 cells, neither a pale blue nor dark red staining is obtained, but rather an intermediate shade, showing prominently dark staining regions in parts. The pause in DNA synthesis observed at mid-S-phase of the diploid Chinese hamster lung fibroblasts seems to be omitted at mid-S-phase of the V-79 cells.

The parental source of heteroploidy in chick embryos determined with chromosomally marked gametes.

Males homozygous for chromosomal translocations were mated to karyologically normal females. The resulting embryos, after incubation for 16-18 h, were prepared for cytogenetic analysis. Of the 62 embryos analysed, 54 (7.8%) were heteroploid or contained a major heteroploid cell line. Each of the 638 normal 2n embryos contained one marker and one normal chromosome, indicating that none arose from gynogenesis. Thirteen homogeneous haploid embryos were identified; 24 embryos were chimaeric 1n/2n and one was 1n/3n. All haploid cell lines contained a marker chromosome indicating androgenetic origins. The 9 homogeneous triploid (3n) embryos and the 3n cell line in a single 1n/3n embryo contained a single marker. All resulted from fertilization by single spermatozoon of eggs that were diploid as a result of suppression of the second meiotic division. The 3n lines of two 2n/3n embryos were derived from other mechanisms. A single homogeneous tetraploid (4n) embryo and the 4n cell lines of three 2n/4n mosaic embryos each contained two marker chromosomes and presumable resulted from failure of cytokinesis in an early cleavage division.