Frequency of chromosome aberrations induced in human peripheral lymphocytes by in vitro 60Co gamma quanta at doses of 1 to 5 Gr. in an analysis of the 1st-division cells at different fixation times

The quantitative analysis of chromosomal aberrations in the first division cells of 50-, 54-, 58-, 52- and 66-hour peripheral blood lymphocytes cultures of healthy donors was performed after irradiation in vitro with 60Co gamma-quantums at doses 1--5 Gy. Cells of the first division were identified by a differential staining of sister chromatid method using 5-bromdeoxyuridine. No significant differences in frequencies of aberrant cells and aberrations of chromosomal type were found between cultures fixed at different times. The distribution of dicentrics in cells did not differ from the Poisson distribution regardless of fixation times and doses. On the basis of these findings it is concluded that chromosomes of human peripheral blood lymphocytes passing the cell cycle at different rates have approximately equal radiosensitivity.     

Genetic determination of fragile-site expression.

To explore genetic effects on the expression of chromosomal fragile sites in vitro, we studied the expression of common fragile sites (c-fra) in cultured lymphocytes of a human chimera (Chi46,XX/46,XY). Since the two cell lines in the chimera share the same environment in vitro and in vivo on cell culture preceding chromosome analysis, differences in the expression of c-fra must be due to genetic factors. The peripheral lymphocytes were cultured in medium 199 and in medium RPMI 1640 with and without aphidicolin. All lesions were localized after GTG-banding and mapped to the human idiogram. In the cultures with aphidicolin the XX cells showed, at high (0.4 microM) APC levels, a significantly higher expression of c-fra than did the XY cells. This difference between cell lineages was not confined to certain individual c-fra; rather it was seen for practically all of them. Therefore, we conclude that there are genetic factors which influence the propensity of c-fra to be expressed. Whether sex is one of these factors, or perhaps even the most important one, still has to be elucidated.     

The cytogenetic, proliferative and viability effects of four bacteriophages on human lymphocytes

Certain bacteriophages have been found in live virus vaccines, while a few others have been associated with disease states. Some of these phages have produced abnormal growth of eukaryotic tissue cultures. For this reason bacteriophages phiX-174, MS2, T2 and an isolate from live virus vaccines, phiV-1, were incubated with human cell cultures for examination of chromosomal effects, cell proliferation and viability. Mitogen-stimulated lymphocytes and human embryonic kidney tissue cultures showed no increase in chromosomal abnormalities for high doses of phage-infected versus control cultures. Tritiated-thymidine uptake, correlated with mitotic indices for phage-treated lymphocyte cultures, indicated a reduction in cell division, while 51-chromium release studies showed no cell death occurring in these cultures. This suggested that inhibition of DNA synthesis was occurring in some cells. The presence of phage in the supernate of cells that were exposed to phage suggested the possibility of phage attachment to the plasma membranes of lymphocytes, which may in turn affect the suppression of DNA synthesis.     

Does proliferation capacity of lymphocytes depend on human blood types?

In vitro human lymphocyte culture methodology is well established yet certain confounding factors such as age, medical history as well as individual’s blood type may potentially modulate in vitro proliferation response. These factors have to be carefully evaluated to release reliable test report in routine cytogenetic evaluation for various genetic conditions, radiation biodosimetry, etc. With this objective, the current study was focused on analyzing the proliferation response of lymphocytes drawn from 90 individuals (21-29 years) with different blood types. The proliferation response was assessed in the cultured lymphocytes by cell cycle, mitotic index (MI), and nuclear division index (NDI) after stimulation with phytohaemagglutinin (PHA). To investigate the toxic effect on proliferation, MI was calculated in representative samples of each blood type were X-irradiated. The results showed that there was no significant difference among the cell cycle phases of lymphocytes in different blood types (P > 0.05). Similarly, both MI and NDI of lymphocytes derived from different blood types also did not show significant difference ( P > 0.05). The extensive interindividual variation within and among the blood types is likely responsible for the lack of significant difference in lymphocyte proliferation. Although spontaneous proliferation efficiency of lymphocytes of different blood types after PHA stimulation was grossly similar, the MI observed after radiation exposure showed a significant difference ( P < 0.05) indicating a differential proliferation response among the blood types. Our results suggest that the blood types did not have any impact on PHA-induced proliferation; however, a specific differential lymphocyte proliferation observed after radiation exposure needs to be considered.     

Sensitivity to X-irradiation of peripheral blood lymphocytes from ageing donors

The proliferation of human blood lymphocytes from ageing donors, responding to concanavalin A, showed greater sensitivity to inhibition by X-rays than similar cells from younger donors. This increased sensitivity was associated with deficiency in repair of X-ray-induced damage to nuclear material, as measured by density in sucrose gradients, and with increased incidence of chromosomal damage following exposure of freshly isolated lymphocytes. There was also an increased frequency of spontaneous chromosomal aberrations in ageing subjects whose lymphocytes were deficient in repair of DNA damage.     

Sex chromosome loss and non-disjunction in women: Analysis of chromosomal segregation in binucleated lymphocytes

Chromosomal lagging and non-disjunction are the main mechanisms of chromosomal malsegregation at mitosis. To date, the relative importance of these two events in the genesis of spontaneous or induced aneuploidy has not been fully elucidated. A methodology based on in situ hybridization with centromeric probes in binucleated lymphocytes was previously developed to provide some insight into this matter. With this method, both chromosomal loss and non-disjunction can be simultaneously detected by following the distribution of specific chromosomes in the nuclei and micronuclei of binucleated cells. In this study, this approach was used for studying the role of chromosomal loss and non-disjunction in the age-related malsegregation of sex chromosomes in females. For this purpose, cultures of cytokinesis-blocked lymphocytes were established from 12 healthy women ranging in age from 25 to 56. The occurrence of malsegregation of X chromosomes in vitro was estimated in binucleated cells that contained four signals, which orginates from the division of normal disomic cells. In this cell population, the frequencies of X chromosome loss and non-disjunction ranged from 0% to 1.69% (mean 0.75%), and from 0.20% to 1.33% (mean 0.57%), respectively. This indicates that both events contribute to malsegregation of X chromosomes in vitro. Moreover, a small but not negligible fraction of binucleated cells with two or six copies of the X chromosome was noticed in all donors. These cells, which are thought to arise from parental monosomic and trisomic types, may indicate the malsegregation of X chromosomes in vivo. The frequency of X chromosome aneuploidy both in vivo and in vitro significantly correlated with the age of donors. Analysis of chromosomal distribution in unbalanced cells demonstrated that both X homologues were frequently involved. The frequency of such multiple events (0.17%) was far greater than that expected by mere chance, indicating a tendency to multiple malsegregation events in the cell population investigated. Finally, parallel analysis of the segregation of chromosomex X and 1 in five of the donors confirmed the greater (about tenfold) susceptibility of X chromosomes to malsegregate compared with autosomes.     

Mechanisms of cell division as regulators of acute immune response

The acute adaptive immune response is complex, proceeding through phases of activation of quiescent lymphocytes, rapid expansion by cell division and cell differentiation, cessation of division and eventual death of greater than 95 % of the newly generated population. Control of the response is not central but appears to operate as a distributed process where global patterns reliably emerge as a result of collective behaviour of a large number of autonomous cells. In this review, we highlight evidence that competing intracellular timed processes underlie the distribution of individual fates and control cell proliferation, cessation and loss. These principles can be captured in a mathematical model to illustrate consistency with previously published experimentally observed data.     

Cell-cycle kinetics of cell lines from patients with chromosomal mosaicism

Lymphocyte cultures from five patients with chromosomal mosaicism (two 47,XY,+21/46,XY, one 47,XX,+21/46,XX, one 45,X/46,XX, and one 47,XXY/46,XY) were studied using sister chromatid differential staining technique for cell kinetic evaluation. Aneuploid and normal cell lines were compared to identify changes in cellular proliferation in vitro that could be related to cellular selective advantage and cell-line-proportion changes occurring with age. Comparison of the percentage of cells in different cell generations in 48, 72, and 96 h-cultures shows no differences between the aneuploid and normal cell lines indicating that cell-cycle kinetics is similar in these cells in vitro.     

Two-compartment model of NK cell proliferation: insights from population response to IL-15 stimulation

NK cells are innate lymphocytes that mediate early host defense against viruses, such as cytomegalovirus. IL-15 is upregulated during viral infections and drives the expansion of NK cells. However, the influence of IL-15 on murine NK cell division and death rates has not been quantitatively studied. Therefore, we developed a series of two-compartment (representing quiescent and dividing NK cell subpopulations) mathematical models, incorporating different assumptions about the kinetic parameters regulating NK cell expansion. Using experimentally derived division and death rates, we tested each model's assumptions by comparing predictions of NK cell numbers with independent experimental results and demonstrated that the kinetic parameters are distinct for nondividing and dividing NK cell subpopulations. IL-15 influenced NK cell expansion by modulating recruitment and division rates to a greater extent than death rates. The observed time delay to first division could be accounted for by differences in the kinetic parameters of nondividing and dividing subsets of NK cells. Although the duration of the time delay to first division was not significantly influenced by IL-15, the recruitment of nondividing NK cells into the replicating subpopulation increased with greater IL-15 concentrations. Our model quantitatively predicted changes in NK cell accumulation when IL-15 stimulation was reduced, demonstrating that NK cell divisional commitment was interrupted when cytokine stimulation was removed. In summary, this quantitative analysis reveals novel insights into the in vitro regulation of NK cell proliferation and provides a foundation for modeling in vivo NK cell responses to viral infections.     

Investigation of genetic markers in a true Hermaphrodite with chi 46,XX/46,XY

Summary We documented a new case of chi 46,XX/46,XY true hermaphroditism substantiated by the evaluation of chromosomal heteromorphism in banded preparations. The patient, a 12-year-old Japanese boy with ambiguous external genitalia, was seen because of abnormal breast development. Surgical exploration showed the right gonad to be an ovotestis and the left gonad to be an ovary. Cytogenetic studies revealed cell admixtures of 46,XX and 46,XY karyotypes in peripheral lymphocytes, skin fibroblasts, and gonadal fibroblasts. From the pedigree studies, the paternal double genetic contributions were evidenced by the differences of sex chromosomes and the blood group types for the ABO and MNSs systems in the two cell lines of the patient. The maternal double genetic contributions were confirmed by the inheritance of Q-fluorescent markers on chromosomes 13 and 22 and by alleles for the Kidd blood group system.     

The cytogenetic,proliferative and viability effects of four bacteriophages on human lymphocytes

Summary Certain bacteriophages have been found in live, virus vaccines, while a few others have been associated with disease states. Some of these phages have produced abnormal growth of eukaryotic tissue cultures. For this reason bacteriophages ϕX-174, MS2, T2 and an isolate from live virus vaccines, ϕV-1, were incubated with human cell cultures for examination of chromosomal effects, cell proliferation and viability. Mitogen-stimulated lymphocytes and human embryonic kidney tissue cultures showed no increase in chromosomal abnormalities for high doses of phage-infected versus control cultures. Tritiated-thymidine uptake, correlated with mitotic indices for phage-treated lymphocyte culture, indicated a reduction in cell division, while 51-chromium release studies showed no cell death occurring in these cultures. This suggested that inhibition of DNA synthesis was occurring in some cells. The presence of phage in the supernate of cells that were exposed to phage suggested the possibility of phage attachment to the plasma membranes of lymphocytes, which may in turn affect the suppression of DNA synthesis. This work was supported by HEW/FDA Grant No. 223-73-1171.     

Mechanisms of regulating cell topology in proliferating epithelia: impact of division plane, mechanical forces, and cell memory

Regulation of cell growth and cell division has a fundamental role in tissue formation, organ development, and cancer progression. Remarkable similarities in the topological distributions were found in a variety of proliferating epithelia in both animals and plants. At the same time, there are species with significantly varied frequency of hexagonal cells. Moreover, local topology has been shown to be disturbed on the boundary between proliferating and quiescent cells, where cells have fewer sides than natural proliferating epithelia. The mechanisms of regulating these topological changes remain poorly understood. In this study, we use a mechanical model to examine the effects of orientation of division plane, differential proliferation, and mechanical forces on animal epithelial cells. We find that regardless of orientation of division plane, our model can reproduce the commonly observed topological distributions of cells in natural proliferating animal epithelia with the consideration of cell rearrangements. In addition, with different schemes of division plane, we are able to generate different frequency of hexagonal cells, which is consistent with experimental observations. In proliferating cells interfacing quiescent cells, our results show that differential proliferation alone is insufficient to reproduce the local changes in cell topology. Rather, increased tension on the boundary, in conjunction with differential proliferation, can reproduce the observed topological changes. We conclude that both division plane orientation and mechanical forces play important roles in cell topology in animal proliferating epithelia. Moreover, cell memory is also essential for generating specific topological distributions.     

Cytogenetic studies using various clastogens in two patients with Werner syndrome and control individuals

Summary Chromosome studies were performed on peripheral lymphocytes from two patients with Werner syndrome and two healthy control individuals to detect spontaneous and/or mutagen-induced chromosomal instability of this disease. Diepoxybutane, isonicotinic acid hydrazide, 4-nitro-quinoline-1-oxide, and bleomycin were used as standard clastogens. While the spontaneous frequency of chromosomal breakage was much higher in lymphocytes from both patients than in the control cells, the basic rate of sister chromatid exchange (SCE) was found to be in the control range. The sensitivity to clastogens of the patients' cells, however, was not substantially increased as compared with the controls if the degree of multiplication of the spontaneous breakage rate or SCE frequency was taken as the basis for comparison. No indication of a greater inhibition of proliferation by the clastogens in the patients' cells than in normal cells was observed using BrdU-labelled lymphocytes. Thus, the lymphocytes from both patients of the present study lacked essential features of the classical chromosome instability syndromes.     

Cellular basis of immunomodulation by cholera toxin in vitro with possible association to the adjuvant function in vivo

Cholera toxin (CT) is a potent oral immunogen that also acts as a strong mucosal adjuvant for immune responses to related as well as unrelated Ag. To elucidate the immunomodulating effects of CT at the cellular level we have examined interactions of CT with APC and with B and T lymphocytes in vitro. CT markedly stimulated the production of IL-1 from APC (mouse peritoneal macrophages or macrophage cell line P388D1) but did not induce Ia-Ag and had marginal, if any, effect in potentiating Ia Ag expression stimulated by rIFN-gamma on these cells. CT had differential effect on T cell proliferation in vitro, usually strongly inhibitory but on Con A-stimulated spleen cells during prolonged (greater than or equal to 5 days) culture or when added on day 4 or later to these cultures up to a two- to three-fold enhancement of proliferation was seen. CT-induced inhibition of T cell proliferation was associated with decreased production of IL-2 and anergy to exogenously added IL-2 despite apparently normal expression of IL-2R. Similar to what was found with T cells LPS-stimulated spleen B cells demonstrated both inhibition and enhancement of proliferation in the presence of CT: in high concentrations (greater than or equal to 10(-8) M) and early in culture (day 3) CT had a strong inhibitory effect on the proliferation of B cells, whereas later (day 6) and/or at lower CT concentrations (10(-9) to 10(-11) M) the proliferation was increased up to 10-fold. The net effect of CT treatment on Ig-production by LPS-stimulated spleen B cells was seen as an enhanced level of IgA and IgG but not IgM in culture supernatants. The differential effects of CT on the cells of the immune system observed in vitro may, singly or in combination, explain the immunostimulatory function of CT.     

Relative ranking of culture media based on proliferation kinetics and spontaneous chromosomal aberrations in PHA-responsive human peripheral blood lymphocytes.

Proliferation kinetics and spontaneous yield of chromosomal aberrations phytohemagglutinin (PHA)-responsive peripheral blood lymphocytes were studied from blood samples collected from 45 individuals in 4 different synthetic media. Except for a significant difference for Eagle's MEM and RPMI 1640, the other media did not show difference for the yield of chromatid or chromosome type of aberrations. Differences were however noticed in the proliferation kinetics (mitotic and proliferative rate indices) of cells among the media used. The study indicated that (i) the intrinsic properties of media which influence proliferation rate and yield of chromosomal aberrations are independent of each other as higher proportion of first division cells do not correspond with higher frequency of chromosomal aberrations, (ii) the amount of free-radical scavengers present in the medium, apart from the genetic make-up of the individuals, may contribute to the spontaneous yield of chromosomal aberrations and (iii) RPMI 1640 medium, which showed higher transformation and faster cycling rate for the lymphocytes, may be considered as medium of choice for analysing two main cytogenetic end-points, chromosomal aberrations and sister chromatid exchanges (SCEs).     

Effect of specific placental proteins--trophoblastic beta 1-glycoprotein chorionic alpha 1-microglobulin--on the proliferation of lymphocytes and malignant fibroblasts in vitro

A study was made of the action of trophoblastic beta 1-glycoprotein (TBG) and chorionic alpha 1-microglobulin (CAG1) on proliferation of malignant fibroblasts (transplanted L line) and on phytohemagglutinin-stimulated lymphocytes. TBG depressed proliferation of the stimulated lymphocytes and transformed fibroblasts (according to 3H-thymidine incorporation). A dose-response dependence was ascertained. CAG1 did not affect cell division. The inhibitory effect of TBG was seen to be reversed or decreased provided the lymphocyte culture was supplemented with CAG1. The decreased inhibitory effect of TBG in the presence of CAG1 was also noted in the L cell culture. It is likely that in vivo protection of intensely proliferating fetal tissues or tumor from the inhibitors is effected just in this way since placental proteins are synthesized both by embryonic and tumorous cells.     

Proliferation of germ cells during gonadal sex differentiation in medaka: Insights from germ cell-depleted mutant zenzai

The proliferation of germ cells becomes sexually dimorphic during gonadal sex differentiation, although the underlying dynamics of this are not well understood in vertebrates. By tracing GFP-labeled germ cells in vivo and analyzing the germ cell-depleted mutant, zenzai, we show that the proliferation and differentiation of germ cells are regulated in a sexually dimorphic manner in the teleost fish medaka. In the undifferentiated gonads, germ cells resume proliferation by slow intermittent division (type I), producing isolated daughter cells. While germ cells in the male gonads continue this mode of proliferation, some germ cell fractions in the female gonads initiate two to four rounds of continuous division (type II), forming cysts of four, eight, or sixteen cells, which subsequently enter meiosis synchronously. Thus, female germ cells become differentiated much earlier than do male germ cells. In the zenzai mutant, a defect in slow intermittent division eventually leads to the depletion of germ cells in the adult gonads in both sexes, despite the fact that cyst-forming division is unaffected. This argues that slow intermittent division is essential for the maintenance of germ cells. The proliferation and differentiation of germ cells are thus important components of gonadal sex differentiation in vertebrates.     

Absence of correlation between the chromosomal radiosensitivity of peripheral blood lymphocytes and stem-cell spermatogonia in mammals

To evaluate the reliability of quantitative extrapolation of radiation-induced chromosomal damage from somatic cells to germ cells, data on the effects of several biological and physical factors on the chromosomal radiosensitivity of blood lymphocytes and stem-cell spermatogonia have been collected from the literature. The results show that most of the factors considered, such as chromosomal constitution, age, genetic constitution, species, sampling time and dose fractionation, had differential effects on the induction of chromosomal aberrations in both systems. These differential effects can easily be explained in terms of the biological differences between in-vitro-stimulated peripheral blood lymphocytes and stem-cell spermatogonia. It is concluded that only direct experiments on germ cells of higher primates and man can be used for a quantitative estimation of human genetic radiation risks arising from structural chromosomal aberrations.     

Cell division and tissue repair following localized damage to the mammalian lung.

Small local wounds on the surface of the mouse lung, produced by cauterization, healed by a typical reparative process involving cell migration and increased cell division in alveolar and bronchial tissues. The local cell division response closely resembled the compensatory cell division response in the same organ which follows unilateral pneumonectomy or unilateral collapse of the lung: initially there was an increase in the rate of DNA synthesis followed by an increased rate of entry into mitosis, both of these functions returning to normal levels within a few days. It is therefore suggested that both types of response are governed by a single regulatory mechanism. The results do not support the view that the rate of cell division is regulated by systemically-circulating mitotic control factors and it is proposed that changes in the cell division rate, both in the reparative and in the compensatory types of response, are determined by local alterations in the concentration of regulatory metabolites. The magnitude of the cell division response was much greater in bronchial than in alveolar tissue, a result which is consistent with the view that new alveolar tissue may be produced by the proliferation and diffentiation of bronchial cells.