Some observations on the localization of mitomycin C-induced aberrations in human lymphocytes

Actively cycling human lymphocytes were treated with mitomycin C for 1 h (1.4 μg/ml) and then grown in medium containing 10 μg/ml bromodeoxyuridine. Serial 5-h colcemid accumulation samples were taken up to 35 h and the air-dried methaphase spreads stained for replication banding. A complete cell-cycle subphasing analysis was made, and classified cells scored for all categories of chromatid-type aberrations and their location.

Inspite of the high dose which produced massive delay and cycle perturbation, there was no evidence for selectively lethality of early-S cells, in fact such cells were in excess. Extreme localization of aberrations to late-replicating (mostly centromeric) regions was found at all subphases and in pre-S cells. This rules out ‘localization by default’ as an explanation for the observed preferential occurence of ‘break points’ in these regions.

The frequency of incomplete intrachanges, low in late S, rises dramaticallyin early S to become maximal in pre-S cells.     

The effect of X-irradiation on cell cycle progression and chromatid aberrations in stimulated human lymphocytes using cohort analysis studies.

Stage sensitivity for the production of chromatid-type aberrations and mitotic delay has been investigated in a stimulated human lymphocyte population, following an absorbed dose of 1.5 Gy 250 kVp X-rays. BrdU replication banding was used to obtain a fine analysis of the cell cycle and to permit cohort analysis. Fluctuations in yield with sample time were found for all aberration categories, but these could not be related simply to either the developmental stage of the cells at time of exposure, or to the time-to-run to metaphase. In general G2 and late S cells had higher aberration yields than early S and pre S cell populations. Mitotic delay and perturbation at this dose extends to all sub-phases of S and is as great, if not greater, in the earliest S cells as it is in G2.     

Chromosomal instability, reproductive cell death and apoptosis induced by O-methylguanine in Mex, Mex and methylation-tolerant mismatch repair compromised cells: facts and models

O⁶-Methylguanine (O⁶-MeG) is induced in DNA by methylating environmental carcinogens and various cytostatic drugs. It is repaired by O⁶-methylguanine-DNA methyltransferase (MGMT). If not repaired prior to replication, the lesion generates gene mutations and leads to cell death, sister chromatid exchanges (SCEs), chromosomal aberrations and malignant transformation. To address the question of how O⁶-MeG is transformed into genotoxic effects, isogenic Chinese hamster cell lines either not expressing MGMT (phenotypically Mex⁻), expressing MGMT (Mex⁺) or exhibiting the tolerance phenotype (Mex⁻, methylation resistant) were compared as to their clastogenic response. Mex⁻ cells were more sensitive than Mex⁺ cells to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)-induced chromosomal breakage, with marked differences in sensitivity depending on recovery time. At early recovery time, when cells out of the first post-treatment mitosis were scored, aberration frequency was about 40% reduced in Mex⁺ as compared to Mex⁻ cells. At later stages of recovery when cells out of the second post-treatment mitosis were analyzed, the frequency of aberrations increased strongly in Mex⁻ cells whereas it dropped to nearly control level in Mex⁺ cells. From this we conclude that, in the first post-treatment replication cycle of Mex⁻ cells, only a minor part of aberrations (<40%) was due to O⁶-MeG whereas, in the second post-treatment replication cycle, the major part of aberrations (>90%) was caused by the lesion. Thus, O⁶-MeG is a potent clastogenic DNA damage that needs two DNA replication cycles in order to be transformed with high efficiency into aberrations. The same holds true for sister chromatid exchanges (SCEs). MNNG is highly potent in inducing SCEs in Mex⁻ cells in the second replication cycle after alkylation. Under these conditions, SCE induction is nearly completely prevented by the expression of MGMT. This is opposed to SCE induction in the first post-treatment replication cycle, where higher doses of MNNG were required to induce SCEs and no protective effect of MGMT was observed. This indicates that SCEs induced in the first replication cycle after alkylation are due to other lesions than O⁶-MeG. In methylation tolerant cells, which are characterized by impaired G–T mismatch binding and MSH2 expression, aberration frequency induced by MNNG was weakly reduced in the first and strongly reduced in the second post-treatment mitoses, as compared to CHO wild-type cells. The results indicate that mismatch repair of O⁶-MeG–T mispairs is decisively involved in O⁶-MeG born chromosomal instability and recombination. We also show that Mex⁺ and methylation tolerant cells are more resistant than Mex⁻ cells with regard to induction of apoptosis, indicating O⁶-MeG to be also an apoptosis-inducing lesion. The data are discussed as to the mechanism of cytotoxicity, aberration and SCE formation in cells treated with a methylating agent.     

Early-replication DNA patterns in a derivative chromosome in a syrian hamster with only 42 chromosomes

From crosses within a 2n = 43 line of Syrian hamsters (Mesocricetus auratus) lacking one derivative (der 11) of an 11;20 reciprocal translocation we have obtained homozygotes with only 42 chromosomes These animals are homozygous deficient (nullisomic) for the centromere and short arm of chromosome 11 and for the bulk of the long arm of chromosome 20. — During cytogenetic studies, we investigated the frequency patterns of early-replicating bands in the surviving derivative (der 20) at two cytologically defined sub-phases of S using short-term fibroblast cultures. These patterns were compared with those observed in the component, untranslocated arms in normal 2n = 44 cells at the same two sub-phases. — Very close agreement was found, indicating that neither the nullisomy, nor the new arm combination has interfered detectably with the pattern or programme of early band replication.     

Secretion of prostaglandin type E (PGE) by tumor cells of Syrian hamsters in in vitro contact with natural killers

The ability of Syrian hamster tumor cells of the same origin but with different degrees of malignancy to secrete prostaglandin E (PGE) was studied following their in vitro contact with Syrian hamster natural killer cells (NK cells). Syrian hamster NK cells were shown to lose significantly cytotoxic activity after their contact with malignant tumor cells. Short-term in vitro contact of malignant tumor cells with human and Syrian hamster NK cells resulted in a rapid PGE secretion into the culturing medium. PGE was determined in the culturing medium, using the biological test, described in the paper, or direct radioimmunoassay. No PGE secretion was observed after the treatment of tumor cells with indomethacin. It is assumed that PGE secretion by malignant tumor cells is one of the mechanisms of their protection against natural killer cells.     

Role of replication time in the control of tissue-specific gene expression.

Late-replicating chromatin in vertebrates is repressed. Housekeeping (constitutively active) genes always replicate early and are in the early-replicating R-bands. Tissue-specific genes are usually in the late-replicating G-bands and therein almost always replicate late. Within the G-bands, however, a tissue-specific gene does replicate early in those cell types that express that particular gene. While the condition of late replication may simply be coincident with gene repression, we review evidence suggesting that late replication may actively determine repression. As mammals utilize a developmental program to Lyonize (facultatively heterochromatinize) whole X chromosomes to a late-replicating and somatically heritable repressed state, similarly another program seems to Lyonize individual replicons. In frogs, all genes begin embryogenesis by replicating during a very short interval. As the developmental potency of embryonic cells becomes restricted, late-replicating DNA gradually appears. This addition to the repertoire of gene control--i.e., repression via Lyonization of individual replicons--seems to have evolved in vertebrates with G-bands being a manifestation of the mechanism.     

Replication time of interspersed repetitive DNA sequences in hamsters

The replication time of 34 hamster genomic DNA segments containing interspersed repeat sequences was determined by probing the cloned segments with nick-translated early- and late-replicating hamster DNA. One-third of these cloned families replicated early, one-third replicated late, and one-third replicated without temporal bias. 19 different inserts from these clones along with the SINE, Alu, and the LINE, A36Fc, were used to probe Southern blots of early- and late-replicating hamster or human DNA. We report long interspersed repeats, LINEs, are selectively partitioned into late-replicating DNA and are often concertedly hypomethylated, while short interspersed repeats, SINEs, are selectively partitioned into early-replicating DNA. For some interspersed repeat families, this partitioning is complete or almost complete. The CCGG frequency is very low in late-replicating DNA. The mammalian chromosome's pattern of early-replicating R-bands and late-replicating G-bands reflects a differential distribution of LINEs and SINEs.     

Comparison between mutagenesis in normal and transformed syrian hamster fibroblasts: Difference in the temporal order of HPRT gene replication

A highly tumorigenic subdiploid cell line, BP6T, derived in our laboratory from Syrian hamster embryo (SHE) cells, is amenable to studies of somatic mutation in vitro. Cellular and biochemical characterization of clonally derived BP6T cells resistant to 6-thioguanine (TG^r) or ouabain (Oua^r) demonstrated these mutants to be similar qualitatively to mutants of SHE cells characterized previously (Barrett et al., 1978). BP6T TG^r mutants resistant to 6-thioguanine are cross-resistant to 8-azaguanine, lack HPRT activity, exhibit a low frequency of reversion and arise spontaneously at a rate of 5 × 10⁻⁷ mutants per cell per generation. BP6T Oua^r mutants were shown to be highly resistant to ouabain-mediated inhibition of ⁸⁶Rb influx, indicating an alteration in the Na⁺/K⁺ ATPase. These studies on the BP6T cell line provide the experimental basis for a comparative study of the mutagenic responses of normal, diploid SHE cells versus those of related, but transformed aneuploid cells. Highly synchronized cultures of these 2 cells were mutagenized by pulse treatment with BrdU during different periods of S phase, followed immediately by near-UV irradiation. The induced mutation frequencies so obtained provided information about the temporal order of replication of genes encoding HPRT and Na⁺/K⁺ ATPase in both SHE and BP6T cells. The temporal pattern of replication of Na⁺/K⁺ ATPase gene loci is similar in both cell types, but the temporal order of replication of the HPRT gene is significantly different between SHE and BP6T cells (mid-late S phase, versus early S phase, resp.). This observed difference emphasizes the caution required in the study of mutagenesis and DNA replication using transformed, aneuploid cells under the assumption that the underlying mechanisms are the same for normal, diploid cells.     

The use of multiple sampling times in in vitro chromosome assays

Asynchronously growing V79 hamster cells were treated with hypotonic solutions of different osmolalities, with ethyl methanesulfonate (EMS) or a combination of these 2 agents. Four different fixation times (8, 10, 14, 18 h) were tested. Hypotonic treatment alone induced chromosomal aberrations (CA), leading to very high frequencies at low osmolalities (50 mOsm/kg H₂O). EMS also induced CA, but a comparison of EMS and hypotonicity revealed that EMS induced fewer aberrations at teh maximum doses tested. A combined treatment produced contradictory results, depending on the sampling time: 8 and 14 h led to a clear elevation of EMS-induced CA by hypotonicity, 10- and 18-h sampling times led to a decrease in the aberration frequency in EMS and hypotonically treated cells.

The observation that the choice of sampling time significantly influences the incidence of chromosomal aberrations is of particular interest for in vitro assays. In this study the 4 different sampling times are not enough to allow meaningful conclusions about additive or synergistic effects of the mutagens tested. These contradictory results are caused, on the one hand, by the very steep dose-response curve after hypotonic treatment and on the other hand by the varying degree of cell-cycle delay after combination treatment. This makes a valid comparison of teh CA frequencies impossible because no 2 sampling times contain the same mixture of cells.     

Preparation of peritoneal cell metaphases of rats, mice and chinese hamsters after mitogenic stimulation with magnesium sulfate and/or aluminum hydroxide

A new cytogenetic system in vivo is presented using peritoneal cells collected from unanesthetized rats, mice and Chinese hamsters, 48 h after an i.p. injection of a mitogen. The test is useful for periodic monitoring of chromosome aberration rates in the investigation of such problems as aging and chronic effects of mutagens and carcinogens.

Among the 2 mitogens tested, aluminum hydroxyde was more efficient than magnesium sulfate in all 3 species.

The rat is considered to be the most suitable animal for this test, in view of its easy handling, high cell yield and favourable response to mitogens. The spontaneous chromosome aberration rate in this animal was found to be comparable with that of bone-marrow cells.     

Chromosomal distribution of the hamster intracisternal A-particle (IAP) retrotransposons

The retrotransposon-like elements of the intracisternal A-particle (IAP) sequences occur in about 900 copies per haploid hamster cell genome. By applying the fluorescent in situ hybridization (FISH) technique and four different, cloned segments of the IAP element as hybridization probes, these elements were found to be distributed in specific patterns over many of the 44 hamster chromosomes. The hybridization patterns were very similar regardless of whether all four probes or only the IAPI probe carrying the long terminal repeat (LTR) region were used. The IAP elements were found most abundantly, though not exclusively, on the short arms of at least 12 of the autosomes. Of the sex chromosomes, the shorter Y chromosome was stained on both arms, and the X chromosome on one arm by the IAP probes. Primary Syrian hamster cells, the established Syrian hamster cell line BHK21, and the adenovirus type 12 (Ad12)-transformed BHK21 cell line T637 yielded very similar results. In Chinese hamster ovary (CHO) or 3T3 mouse cells, signals could not be elicited by FISH using the Syrian hamster IAP probes. On Southern blots, the DNAs from these cell lines hybridized very weakly, if at all, to the IAP sequences. Thus, IAP sequences were retroposed after Syrian hamster and mouse or Syrian and Chinese hamsters had diverged in evolution.     

Neoplastic transformation of cultured Syrian hamster embryo cells by DNA of herpes simplex virus type 2.

Syrian hamster embryo cells were transformed to a neoplastic phenotype after exposure to herpes simplex virus type 2 (S-1) DNA at concentrations (less than or equal to 0.01 microgram per 60-mm dish) at which infectivity was no longer demonstrable. Transformed cells manifested in vitro phenotypic properties characteristic of the neoplastic state, expressed herpes simplex virus-specific antigens, and induced invasive tumors in vivo. Transfection and transformation of Syrian hamster embryo cells with herpes simplex virus type 2 DNA or its fragments is a suitable system for investigating the structure and function of herpes simplex virus-transforming gene(s).     

Early induction of polyfunctional simian immunodeficiency virus (SIV)-specific T lymphocytes and rapid disappearance of SIV from lymph nodes of sooty mangabeys during primary infection

Although the cellular immune response is essential for controlling SIV replication in Asian macaques, its role in maintaining nonpathogenic SIV infection in natural hosts such as sooty mangabeys (SM) remains to be defined. We have previously shown that similar to rhesus macaques (RM), SM are able to mount a T lymphocyte response against SIV infection. To investigate early control of SIV replication in natural hosts, we performed a detailed characterization of SIV-specific cellular immunity and viral control in the first 6 mo following SIV infection in SM. Detection of the initial SIV-specific IFN-γ ELISPOT response in SIVsmE041-infected SM coincided temporally with a decline in peak plasma viremia and was similar in magnitude, specificity, and breadth to SIVsmE041-infected and SIVmac239-infected RM. Despite these similarities, SM showed a greater reduction in postpeak plasma viremia and a more rapid disappearance of productively SIV-infected cells from the lymph node compared with SIVmac239-infected RM. The early Gag-specific CD8(+) T lymphocyte response was significantly more polyfunctional in SM compared with RM, and granzyme B-positive CD8(+) T lymphocytes were present at significantly higher frequencies in SM even prior to SIV infection. These findings suggest that the early SIV-specific T cell response may be an important determinant of lymphoid tissue viral clearance and absence of lymph node immunopathology in natural hosts of SIV infection.     

Gestation stage-specific frequency of adipogenic cells in Syrian hamster cell cultures

High frequencies (up to 50%) of spontaneous adipocyte differentiation are observed in cultures of 9 day gestation Syrian hamster embryos (E9 cells) within six to eight population doublings after primary culture. This is in contrast to the absence of adipogenic cells in primary cultures derived from later gestation age Syrian hamster tissue. In addition, E9 primary cultures contain a transient subpopulation of presumptive mesenchymal stem or progenitor cells that lack density dependent inhibition of growth [contact-insensitive (CS-) cells]. Analysis of the temporal pattern of expression of the CS- and adipocyte phenotypes during the proliferative life span of E9 cells demonstrates that maximal expression of the CS- phenotype precedes maximal expression of adipocyte differentiation. In addition, lipid accumulation appears to occur primarily, if not exclusively, in the contact-sensitive (CS+) cells that are derived from CS- cells. These observations suggest that primary E9 cultures contain either adipoblasts or primordial mesenchymal cells that become determined to the adipocyte lineage early during the in vitro life span of the cultures, and that the CS- phenotype may be a marker for these earlier developmental cell stages.     

Uninterrupted DNA synthesis during S-phase in untransformed diploid hamster fibroblasts

Untransformed Syrian hamster fibroblasts in exponential growth were exposed to a pulse of [³H]-thymidine for 5 min, followed immediately by bromodeoxyuridine, and serial samples were taken up to 16 h. Preparations were autoradiographed and stained for replication banding. No cell with replication bands was found without significant [³H]-thymidine uptake, although the extent of uptake varied between sub-phases of S. Thus there is no indication of a total cessation of synthesis at any period during S-phase.     

Further characterization of a CD99-related 21-kDa transmembrane protein (VAP21) expressed in Syrian hamster cells and its possible involvement in vesicular stomatitis virus production

The VAP21, a CD99-related 21-kDa transmembrane protein, was first detected in the enveloped virions that were grown in a Syrian hamster-derived cell line, BHK-21 (Sagara et al., 1997; Yamamoto et al., 1999). We further tried to elucidate the nature and properties of VAP21. The VAP21 was detected in various organs of the Syrian hamster as well as in the Syrian hamster-derived cell lines (BHK-21 and HmLu-1). We could not detect the VAP21 antigen in other cell lines derived from other animal species we examined, including a Chinese hamster (CHO-K1), mouse (neuroblastoma C1300, clone NA), dog (MDCK), monkey (COS-7), and human (HeLa, HepG2). We tried to introduce the VAP21 gene into VAP21-negative cell lines using a tetracycline-regulated gene expression system. All of our trials, however, resulted in failure to establish stably positive inducible cell lines. To the contrary, we could easily establish the VAP21-overexpressing cell lines from the Syrian hamster cell lines, which were successfully grown and maintained without any loss of VAP21 expression even under the induced culture conditions. In such VAP21-overexpressing cells, production of the vesicular stomatitis virus (VSV) was increased several-fold, while suppression of the VAP21 expression resulted in reducing the VSV yields. From these results, we conclude that the VAP21 is a physiologically active cell membrane component of some animal species including the Syrian hamster, and might positively be involved in the VSV replication.     

The relevance of caffeine post-treatment to SCE incidence induced in Chinese hamster cells

The influence of caffeine post-treatment on sister-chromatid exchanges (SCE) and chromosomal aberration frequencies on Chinese hamster cells exposed to a variety of chemical and physical agents followed by bromodeoxyuridine (BrdUrd) was determined. After 2 h treatment, N-methyl-N′-nitrosoguanidine (MNNG) and cis-platinum(II)diamine dichloride (cis-Pt(II)) induced a 7- and 6-fold increase in SCE, respectively, while 4-nitroquinoline-1-oxide (4NQO), methyl methanesulfonate (MMS), proflavine, and N-hydroxyfluorenylacetamide (OH-AAF) caused a 2–3-fold increase in SCE compared to controls treated with BrdUrd alone. Ultraviolet light doubled the number of SCE. The lowest increase of SCE was obtained with bleomycin and X-irradiation. Caffeine post-treatment caused a statistically significant increase in the frequency of SCE induced by UV- and X-irradiation as well as by 4NQO and MMS but did not alter the number of SCE induced by MNNG, cis-Pt(II), proflavine, OH-AAF, and bleomycin.

Caffeine post-treatment increased the number of cells with chromosomal aberrations induced by MNNG, cis-Pt(II), UV, 4NQO, MMS, and proflavine. With the exception of proflavine, these agents are dependent on DNA and chromosome replication for the expression of the chromosomal aberrations. Caffeine enhancement of cis-Pt(II) chromosomal aberrations occurred independently of the time interval between treatment and chromosome preparations. Chromosomal damage produced by bleomycin and X-irradiation, agents known to induce chromosomal aberrations independent of “S” phase of the cell cycle, as well as the damage induced with OH-AAF was not influenced by caffeine post-treatment.

The enhancement by caffeine, an inhibitor of the gap-filling process in post-replication repair, of chromosomal aberrations induced by “S” dependent agents, is consistent with the involvement of this type of repair in chromosomal aberration formation. The lack of inhibition of SCE frequency by caffeine indicates that post-replication repair is probably not important in SCE formation.