Syrian Hamster Embryonic Cells Research Articles

The Syrian Hamster Embryo (SHE) Low pH Cell Transformation Assay

The Syrian hamster embryonic (SHE) cell transformation assay (CTA) at pH 6.7 can determine the ability of a test article to induce morphological transformation (MT) in cultured SHE cells. The assay uses SHE cells prepared at gestation day ∼13 and frozen. Target cells are seeded onto a layer of feeder cells (X-ray irradiated SHE cells) and treated with test article for 24 hr or 7 days. After a growth period of 7 days, the cells are fixed, stained, and evaluated for MT. Normal colonies typically contain a monolayer of cells with an organized, often flowing, pattern of growth and minimal cell criss-crossing at a confluent density. Transformed colonies contain randomly oriented, stacked cells, with cell criss-crossing throughout the colony. The transformed cells are frequently more basophilic than their normal counterparts, with increased nuclear/cytoplasmic ratios. This assay provides a valuable tool for evaluating the carcinogenic potential of a test article.

Reversible phenotype and lack of direct link to immortalization of Syrian hamster embryonic cells obtained from so-called transformed colonies

The short-term colony transformation assay employing Syrian hamster embryonic (SHE) cells has been widely used as a simple method for detection of chemical and physical carcinogens. However, little investigation has been done on the biological properties of the early transformed colony (ETC: colony characterized by piling up and criss-cross pattern of growth) itself. This study was performed to examine the properties of these colonies. Secondary or tertiary cultures of SHE cells were treated with benzo[a]pyrene or N-methyl-N’-nitro-N-nitrosoguanidine. In total, 37 ETCs and 17 normal colonies (NCs) were cloned and analyzed. Obtained results were as follows: (1) Stability of transformed morphology; immediately after cloning, the cells from 3/37 of the ETCs maintained their transformed phenotype, but all cells from other ETCs (34/37) showed flat or well-oriented morphology. Thus, the “transformed” morphology of more than 90% of the ETCs was reversible. (2) Chromosome abnormality; 3/15 of the clones from ETCs were hypo diploid or tetraploid, while the others (12/15) were normal diploid immediately after cloning. (3) Immortalization; up to about one month after cloning, most of the clones (from transformed or normal colonies) could be subcultured at 1:2 or 1:4 split ratio per week, but thereafter all the clones ceased growing. After about a one month or longer latency, 6/37 of the clones from ETCs and 4/17 of the clones from NCs restarted growing and acquired immortality. That is, there was no significant difference in the frequency of immortalization between ETCs and NCs. Thus, from the present experiment, there was no direct evidence that ETC correlates to acquisition of immortality or tumorigenesis. Further experiments (e.g. comparison of gene expression profiles between cells from transformed and normal colonies using microarray) would be required to give a logical meaning to this short-term transformation assay.

Influence of the sampling time on chromosomal aberrations at G 2 phase in Syrian hamster embryonic cells irradiated with different types of radiation

Purpose : To investigate the time-course of chromosomal aberrations following radiations of differing LET. Materials and Methods : Syrian hamster embryonic cells were irradiated with nitrogen ions (LET infinity = 530 keV/ μ m) and helium-ions (LET infinity = 36 and 77 keV/ μ m), also 137 Cs γ-rays as a reference radiation. The frequency of chromatid-type aberrations was determined after 0–6 h incubation in a 5% CO 2 incubator at 37°C. Results : The amount of chromosomal damage per cell for nitrogen ions detected immediately after irradiation was lower than induced by 137 Cs gamma-rays. In contrast, helium ions were more effective than gamma rays in inducing chromatid type damage. The RBE values for the nitrogen-ion beams were 0.45 for gaps, 0.43 for deletions and 0.20 for exchanges. For helium-ion beams, the RBE values for the 36 keV/mum beams and the 77keV/ μ m beams were 1.2 and 1.5 for gaps, 1.3 and 2.1 for deletions, and 1.5 and 1.9 for exchanges, respectively. The frequency of cells with chromosomal damage following exposure to γ-rays and helium-ion beams showed a downward trend with increasing incubation period. In contrast, in the case of nitrogen-ion beams, there was an increase with the incubation period. Conclusions : The results show that it is possible to underestimate chromosomal damage for different types of radiation by scoring aberrations at a single fixed sampling time.

The Localization of Ultraviolet-Induced Excision Repair in the Nucleus and the Distribution of Repair Events in Higher Order Chromatin Loops in Mammalian Cells

ABSTRACT Several lines of evidence indicate that eukaryotic DNA is arranged in highly supercoiled domains or loops, and that the repeating loops are constrained by attachment to a nuclear skeletal structure termed the nuclear matrix. Active genes are transcribed at the nuclear matrix and during replication the loops are reeled through fixed matrix-associated replication complexes. We have investigated whether the repair of DNA damage also occurs in the nuclear matrix compartment. Biochemical analysis of confluent normal human fibroblasts, ultraviolet (u.v.)-irradiated with 30 J m−2 and post-u. v. incubated in the presence of hydroxyurea, did not show any evidence for the occurrence of repair synthesis at the nuclear matrix either 30 min or 13 h after irradiation. Autoradiographic visualization of repair events in single DNA halo-matrix structures confirmed the biochemical observations. At a biologically more relevant dose of 5 J m−2 repair synthesis seems to initiate at the nuclear matrix, although only part of the total repair could be localized there. In u.v.-irradiated (30 J m−2) normal human fibroblasts post-u.v. incubated in the presence of hydroxyurea and arabinosylcytosine for 2h, multiple single-stranded regions are generated in a DNA loop as a result of the inhibition of the excision repair process. Different biochemical approaches revealed that most of the single-stranded regions are clustered, indicating that the repair process itself is non-random or that domains in the chromatin are repaired at different rates. Preferential repair of certain domains in the chromatin was shown to occur in xeroderma pigmentosum cells of complementation group C (XP-C). In XP-C cells these domains are localized near the attachment sites of DNA loops at the nuclear matrix. In contrast, xeroderma pigmentosum cells of complementation group D as well as Syrian hamster embryonic cells with limited excision-repair capacities, revealed a random distribution of repair events in DNA loops. The preferential repair of matrix-associated DNA in XP-C cells may be related partly to repair of transcriptionally active DNA and this may account for the ability of XP-C cells, in contrast to XP- D cells, to recover u.v.-inhibited synthesis of DNA and RNA.

Distribution of u.v.-induced repair events in higher-order chromatin loops in human and hamster fibroblasts.

The repair of u.v.-induced damage in human and rodent cells was investigated at the level of DNA loops attached to the nuclear matrix. After 2 h post-u.v. incubation, DNase I digestion studies revealed a 3- to 4-fold enrichment of repair-labeled DNA at the nuclear matrix in four xeroderma pigmentosum cell strains belonging to complementation group C. This non-random distribution was not affected by treatment with sodium butyrate. In other cells with limited excision repair, i.e. two xeroderma pigmentosum cell strains of complementation group D and Syrian hamster embryonic cells, as well as in HeLa cells and normal human fibroblasts, no enrichment of repair-labeled DNA at the nuclear matrix was observed. Visualization of repair events in DNA loops by autoradiography of DNA halo-matrix structures confirmed the biochemical observations. The presence or absence of preferential repair of nuclear matrix-associated DNA paralleled the presence or absence of inhomogeneity in the distribution of T4 endonuclease-V-sensitive sites. A detailed analysis of repair events in xeroderma pigmentosum cells of complementation group C showed that after 2 h post-u.v. incubation, repair events were found at both attachment sites in a limited number of loops and that large domains of loops were not subjected to repair.

Increase during passage in culture in susceptibility of syrian hamster embryonic cells to induction of chromosome aberrations by 7,12-dimethylbenz(a)-anthracene

Studies were made on chromosome aberrations in Syrian hamster embryonal fibroblasts (SHEF) at early and late passages in culture. Aberrations were induced with a potent indirect clastogen, 7,12-dimethylbenz(a)anthracene (DMBA). Results showed that cells at late passages were much more susceptible to this chemical than cells at early passages: the frequency of aberrant metaphases was usually < 20% at the 2nd passage, but it gradually increased with increase in the number of culture passages, reaching 50% or more at the 8th passage in one experiment and 6th–7th passage in another. Induction of chromosome aberrations was also examined in V79 cells co-cultured with hamster cells at early and late passages. V79 cells are known to have no drug-metabolizing enzyme activity. Results demonstrate that the frequency of chromosome aberrations of V79 cells was much greater in the presence of hamster cells at late passages than in the presence of cells at early passages.

Chromosomal aberration, mutation and morphological transformation of Syrian hamster embryonic cells after exposure to methylnitrosocyanamide

Hamster embryonic fibroblasts were treated directly with various concentrations of methylnitrosocyanamide (MNC), a nitrosated product of methylguanidine (MG) or N-methyl- N′-nitro- N-nitrosoguanidine (MNNG). Then they were examined for chromosomal aberrations, morphological transformation and mutations resistant to 8-azaguanine (8AG) and 6-thioguanine (6TG). Direct treatment with 2 to 10 × 10 −6 M MNC caused a marked, dose-dependent appearance of 8AG- and 6TG-resistant mutations. The ability of MNC to induce mutations was similar to that of MNNG. Cultured embryonic fibroblasts in metaphase plates also showed a marked dose-dependent increase in chromosomal aberrations within 24 h after direct treatment with MNC of MNNG. Moreover, MNC and MNNG caused similar rates of morphological transformation.

The attachment and penetration of T7 [formula omitted] in Syrian hamster embryonic cells

Bacteriophage T7 DNA can penetrate Syrian hamster embryonic cells after a mandatory initial pretreatment with DEAE-dextran. In 3 h an extracellular complex between T7 DNA and the cell monolayer is formed which is equivalent to 10 5 T7 genomes per cell. During the ensuing 24–48 h of cell growth, an average of 10 2–10 3 T7 genomes are transported to the nucleus in 90% of the cells of the culture.