Regulatory substances produced by lymphocytes. V. Production of inhibitor of DNA synthesis (IDS) by proliferating T lymphocytes.

The conditions neccessary for production of inhibitor of DNA synthesis (IDS) by rat lymphocytes were investigated.In concanavalin A (Con A)-stimulated lymph node cell (LNC) cultures, IDS production was not detected in the culture supernatant during the first 24 hr, and it increased gradually after that to reach a maximum at 3 to 4 days.When the cells were pretreated with mitomycin C, IDS was not produced, suggesting that DNA synthesis of LNC or a LNC subpopulation is necessary for IDS production. In contrast, Con A-stimulated spleen cells priduced a high level of IDS within 24 hr, and its production fell off sharply thereafter. Con A-stimulated rat thymocytes also produced IDS reaching a maximum at 2 to 3 dyas. However, thymus cells from rats treated with hydrocortisone 48 hr previously did not produce IDS. This finding implies that cortisol-sensitive (cortical) thymocytes are capable of producing IDS and cortisol-resistant (medullary) thymocytes are not. IDS production by lymphoblasts was proportional to cell number and unaffected eith by cell density (1 to 10 x 106/ml) or by the concomitant presence of normal cells from spleen, lymph node, or thymus. Thus Con A-stimulated cells, after becoming blasts, appear to produce IDS automatically wihtout affecting or being affected by other cells. Both spleen and thymus cells from rats injected with a large dose of antigen (ovalbumin, 100 mg, i.p.) 24 hr in advance produced substantial amounts of IDS in culture within 24 hr in the absence of mitogen or additional antigen, but not the cells from rats injected with an immunizing dose (1 mg) of the same antigen. The cells producing IDS in the spleen were shown to be adherent to glass wool, and those in the thymus were partially so. IDS production by antigen-stimulated spleen cells was abrogated by injecting rats with bromodexyuridine (BUdR) at 0 and 12 hr after the ovalbumin. These findings suggest that a subpopulation ofadherent spleen cells (possibly resembling cortical thymocytes), which begins to proliferate within a few hours after a large dose of systemic antigen, produces IDS. This may account for increased nonspecific suppressor activity observed at the same time.     

用双参数流式细胞光度术(FCM)研究阿克拉霉素对L_(1210)白血病细胞周期的影响

本文用双参数FCM技术,对同一个细胞的DNA和RNA含量进行相关测量,比较了ACM B对小鼠L_(1210)白血病细胞周期和RNA含量的影响.结果发现在一次给药后8小时可导致早、中期S的积累,并抑制S期细胞的DNA合成;到24小时DNA合成恢复正常,并进入G_2期,但由于G_2期细胞进入M期受阻,造成G_2期细胞的积累,这时被阻断在G_2期的细胞RNA含量显著增加,形成正不平衡生长,而给药剂量较大的实验组(1/1.5LD_(50))S期细胞的RNA含量不随着DNA含量的增加而增加,形成负不平衡生长,ACM A和ACM B对体内Li_(210)细胞周期作用相同.     

5C6-F4, a novel 100,000-dalton rat lymphocyte activation antigen defined by monoclonal antibody

Con A-activated rat thymocytes were used to immunize mice, and immune spleen cells were fused with NS/1 myeloma cells. One clone, designated 5C6-F4, reacted strongly with Con A-activated rat thymocytes and some LPS-activated rat spleen cells but not with normal thymocytes, spleen cells, or bone marrow cells of rat origin. The 5C6-F4 did not react with Con A-activated thymocytes of mouse origin. Immunoprecipitation of 5C6-F4 antigen from surface-iodinated Con A-activated rat thymocytes or LPS-activated rat spleen cells revealed its m.w. to be approximately 100,000. The kinetic studies of the expression of 5C6-F4 antigen revealed that 5C6-F4 antigen was detectable at 6 hr after Con A stimulation of rat spleen cells, whereas IL 2 receptor (IL 2R) was detectable at 12 hr. The appearance of 5C6-F4 antigen and IL 2R precede the onset of DNA synthesis of Con A-activated spleen cells. Thus, 5C6-F4 antigen is classified as early activation antigen. The 5C6-F4 inhibits the lymphocyte proliferation induced by mitogen and the IL 2-driven rat T cell proliferation. Sequential immunoprecipitation study as well as binding inhibition study indicated that the 5C6-F4 antigen is distinct from IL 2R molecule. The 5C6-F4 antigen appears to be a novel rat lymphocyte activation antigen that exhibits immunoregulatory function and also may serve as a useful marker of T cell activation.     

Phosphoinositide metabolism and the calcium response to concanavalin A in S49 T-lymphoma cells. A comparison with thymocytes.

Comparisons were made between transformed S49 T-lymphoma cells and normal murine thymocytes in their polyphosphoinositides, inositol polyphosphates and cytosolic free calcium concentrations ([Ca2+]i), and the effects of the T-cell mitogen concanavalin A (Con A) on these properties. 1. The ratios of the polyphosphoinositides to phosphatidylinositol in both exponential-phase S49 cells and mitogen-stimulated thymocytes (G1 phase) were greater than in quiescent (G0-phase) thymocytes. 2. In response to Con A, the amount of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) in S49 cells decreased slightly (17% in 30 min), and this was sufficient to account for the small amounts of inositol phosphates that accumulated. In contrast, it has been shown previously that Con A stimulates a rapid resynthesis of PtdInsP2 in thymocytes and the amounts of inositol phosphates released rapidly exceed the steady-state amount of the PtdInsP2 precursor [Taylor, Metcalfe, Hesketh, Smith & Moore (1984) Nature (London) 312, 462-465]. 3. The [Ca2+]i did not differ significantly in S49 cells and thymocytes before the addition of Con A, and the increases in [Ca2+]i in response to Con A were similar in both types of cell. 4. The [Ca2+]i increase in response to Con A was inhibited by similar concentrations of intracellular cyclic AMP (2-10 microM) in S49 cells and thymocytes, suggesting that similar regulatory mechanisms act on this response in both types of cell. The data demonstrate that the basal [Ca2+]i and phosphoinositide metabolism is similar in both the normal cells and their transformed counterparts. In addition, they suggest that the activated Con A receptors generate very similar signals in the two cell types, and that any perturbations of primary signal transduction to the secondary phosphoinositide and [Ca2+]i responses in the S49 phenotype are quantitative rather than qualitative.     

Cell Cycle Kinetics of Aerated, Hypoxic and Re-Aerated Cells In Vitro Using Flow Cytometric Determination of Cellular Dna and Incorporated Bromodeoxyuridine

Abstract. The effects of extreme hypoxia on cell cycle progression were studied by simultaneous determination of DNA and bromodeoxyuridine (BrdU) contents of individual cells. V79-379A cells were pulse-labelled with BrdU (1 μM, 20 min, 37°C) and then incubated for up to 12 hr in BrdU-free medium under either aerated or extremely hypoxic conditions. After the incubation interval (0-12 hr), the cells were trypsinized and fixed in 50% EtOH. Propidium iodide and a fluorescein-labelled monoclonal antibody to BrdU were then used to quantify DNA content and incorporated BrdU, respectively. Measurements in individual cells were made by simultaneous detection of green and red fluorescence upon excitation at 488 nm using flow cytometry. Bivariate analysis revealed progression of BrdU-labelled cells in aerated cultures out of S phase, into G₂ and cell division, with halving of mean fluorescence, and back into S phase by approximately 9 hr after the BrdU pulse. Hypoxia immediately arrested cells in all phases of the cell cycle. Both the DNA distribution and the bivariate profile of cells that were fixed from 2 to 12 hr after induction of hypoxia were identical to the 0 hr controls. the percent of cells with green fluorescence in a mid-S phase window remained 100% and the mean fluorescence of these cells remained at control (0 hr) levels. This indicates that, under hypoxic conditions, cells were moving neither into nor out of S phase. Cultures that had been hypoxic for 12 hr exhibited an increasing rate of BrdU uptake with time after re-aeration. Re-aerated cells were able to complete or initiate DNA synthesis, but their rates of progression through the cell cycle were markedly reduced. A large fraction of cells appeared unable to divide up to 12 hr following release from hypoxia.     

Cell cycle kinetics of aerated, hypoxic and re-aerated cells in vitro using flow cytometric determination of cellular DNA and incorporated bromodeoxyuridine

The effects of extreme hypoxia on cell cycle progression were studied by simultaneous determination of DNA and bromodeoxyuridine (BrdU) contents of individual cells. V79-379A cells were pulse-labelled with BrdU (1 microM, 20 min, 37 degrees C) and then incubated for up to 12 hr in BrdU-free medium under either aerated or extremely hypoxic conditions. After the incubation interval (0-12 hr), the cells were trypsinized and fixed in 50% EtOH. Propidium iodide and a fluorescein-labelled monoclonal antibody to BrdU were then used to quantify DNA content and incorporated BrdU, respectively. Measurements in individual cells were made by simultaneous detection of green and red fluorescence upon excitation at 488 nm using flow cytometry. Bivariate analysis revealed progression of BrdU-labelled cells in aerated cultures out of S phase, into G2 and cell division, with halving of mean fluorescence, and back into S phase by approximately 9 hr after the BrdU pulse. Hypoxia immediately arrested cells in all phases of the cell cycle. Both the DNA distribution and the bivariate profile of cells that were fixed from 2 to 12 hr after induction of hypoxia were identical to the 0 hr controls. The percent of cells with green fluorescence in a mid-S phase window remained 100% and the mean fluorescence of these cells remained at control (0 hr) levels. This indicates that, under hypoxic conditions, cells were moving neither into nor out of S phase. Cultures that had been hypoxic for 12 hr exhibited an increasing rate of BrdU uptake with time after re-aeration. Re-aerated cells were able to complete or initiate DNA synthesis, but their rates of progression through the cell cycle were markedly reduced. A large fraction of cells appeared unable to divide up to 12 hr following release from hypoxia.     

Evidence for a Relationship Between Equine Abortion (Herpes) Virus Deoxyribonucleic Acid Synthesis and the S Phase of the KB Cell Mitotic Cycle

Autoradiographic analyses of deoxyribonucleic acid (DNA) synthesis in randomly growing KB cell cultures infected with equine abortion virus (EAV) suggested that viral DNA synthesis was initiated only at times that coincided with the entry of noninfected control cells into the S phase of the cell cycle. Synchronized cultures of KB cells were infected at different stages of the cell cycle, and rates of synthesis of cellular and viral DNA were measured. When cells were infected at different times within the S phase, viral DNA synthesis was initiated 2 to 3 hr after infection. However, when cells in G1 and G2 were infected, the initiation of viral DNA synthesis was delayed and occurred only at times corresponding to the S phase. The times when viral DNA synthesis began were independent of the time of infection and differed by as much as 5 hr, depending on the stage of the cell cycle at which cells were infected. Viral one-step growth curves were also related to the S phase in a manner which indicated a relationship between the initiation of viral DNA synthesis and the S phase. These data support the concept that initiation of EAV DNA synthesis is dependent upon some cellular function(s) which is related to the S phase of the cell cycle.     

Monoclonal antibodies that inhibit activation and proliferation of lymphocytes. I. Expression of the antigen on monocytes and activated lymphocytes

It has been shown previously that HBJ127 and HBJ98 monoclonal antibodies raised against a human bladder cancer cell line, and B3 monoclonal antibody against a rat bladder cancer cell line recognized unique cell surface antigens abundant in proliferating cells of the corresponding species. Distribution of the antigens and kinetics of the appearance on human and rat lymphoid cells were examined by means of flow cytometry. Rat macrophages and human peripheral blood monocytes were stained strongly with the B3 and HBJ127 monoclonal antibodies, respectively. With regard to lymphocytes, the expression of the B3-defined antigen on rat lymphocytes was found to have a negative correlation with the maturation of the lymphocytes; the antigen was most abundant in bone marrow cells, less abundant in thymocytes, and least abundant in spleen, lymph node, and peripheral blood lymphocytes. Similarly, the HBJ127-defined antigen on human peripheral lymphocytes was negligible. On activation with Con A or alloantigens, however, both rat and human T lymphocytes did strongly express these antigens. Activation of human or rat B cells with lipopolysaccharide also resulted in the augmented expression of these antigens. Kinetics studies revealed that the antigen expression was readily manifested within 12 hr on activation of rat or human T cells with Con A, was augmented progressively with culture time, and reached a plateau within 36 hr. This somewhat earlier appearance of these antigens apparently preceded the manifestations of the IL 2 receptor (Tac antigen) and the augmented DNA synthesis. The B3-defined antigen on Con A-stimulated T cells was more rich on the lymphocytes in S and G2/M phases than those in G1 phase, and the expression was not significantly affected by the addition of hydroxyurea, but was moderately inhibited by the addition of sodium butylate. These results suggest that the appearance and expression of the B3-defined antigen and probably also those of the HBJ127/HBJ98-defined antigen are correlated with lymphocyte activation and subsequent progression through the cell cycle.     

Mode of estrogen action on cell proliferation in CAMA-1 cells: II. Sensitivity of G1 phase population

The mammary cancer cell line CAMA-1 synchronized at the G1/S boundary by thymidine block or at the G1/M boundary by nocodazole was used to evaluate 1) the sensitivity of a specific cell cycle phase or phases to 17 beta-estradiol (E2), 2) the effect of E2 on cell cycle kinetics, and 3) the resultant E2 effect on cell proliferation. In synchronized G1/S cells, E2-induced 3H-thymidine uptake, which indicated a newly formed S population, was observed only when E2 was added during, but not after, thymidine synchronization. Synchronized G2/M cells, enriched by Percoll gradient centrifugation to approximately 90% mitotic cells, responded to E2 added immediately following selection; the total E2-treated population traversed the cycle faster and reached S phase approximately 4 hr earlier than cells not exposed to E2. When E2 was added during the last hour of synchronization (ie, at late G2 or G2/M), or for 1 hr during mitotic cell enrichment, a mixed response occurred: a small portion had an accelerated G1 exit, while the majority of cells behaved the same as controls not incubated with E2. When E2 addition was delayed until 2 hr, 7 hr, or 12 hr following cell selection, to allow many early G1 phase cells to miss E2 exposure, the response to E2 was again mixed. When E2 was added during the 16 hr of nocodazole synchronization, when cells were largely at S or possibly at early G2, it inhibited entry into S phase. The E2-induced increase or decrease of S phase cells in the nocodazole experiments also showed corresponding changes in mitotic index and cell number. These results showed that the early G1 phase and possibly the G2/M phase are sensitive to E2 stimulation, late G1, G1/S, or G2 are refractory; the E2 stimualtion of cell proliferation is due primarily to an increased proportion of G1 cells that traverse the cell cycle and a shortened G1 period, E2 does not facilitate faster cell division; and estrogen-induced cell proliferation or G1/S transition occurs only when very early G1 phase cells are exposed to estrogen. These results are consistent with the constant transition probability hypothesis, that is, E2 alters the probability of cells entering into DNA synthesis without significantly affecting the duration of other cell cycle phases. Results from this study provide new information for further studies aimed at elucidating E2-modulated G1 events related to tumor growth.     

Macrophage-mediated cytostatic activity blocks lymphoblast cell cycle progression independently in both G1 phase and S phase

Recent work has shown that macrophage-mediated cytostatic activity inhibits cell cycle traverse in G1 and/or S phase of the cell cycle without affecting late S, G2, or M phases. The present report is directed at distinguishing between such cytostatic effects on G1 phase or S phase using the accumulation of DNA polymerase alpha as a marker of G1 to S phase transition. Quiescent lymphocytes stimulated with concanavalin A undergo a semisynchronous progression from G0 to G1 to S phase with a dramatic increase in DNA polymerase alpha activity between 20 and 30 hr after stimulation. This increase in enzyme activity was inhibited, as was the accumulation of DNA, when such cells were cocultured with activated murine peritoneal macrophages during this time interval. However, if mitogen-stimulated lymphocytes were enriched for S-phase cells by centrifugal elutriation and cocultured with activated macrophages for 4-6 hr, DNA synthesis was inhibited but the already elevated DNA-polymerase activity was unaffected. Similar results were obtained when a virally transformed lymphoma cell line was substituted as the target cell in this assay. These results show that both G1 and S phase of the cycle are inhibited and suggest that inhibition of progression through the different phases may be accomplished by at least two distinct mechanisms.     

Regenerative proliferation of mouse epidermal cells following adhesive tape stripping. Micro-flow fluorometry of isolated epidermal basal cells combined with 3H-TdR incorporation and a stathmokinetic method (colcemid).

The proliferating cells of mouse epidermis (basal cells) can be separated from the non-proliferating cells (differentiating cells) Laerum, 1969) and brought into a monodisperse suspension. This makes it possible to determine the cell cycle distributions (e.g. the relative number of cells in the G1, S and (G1 + M) phases of the cell cycle) of the basal cell population by means of micro-flow fluorometry. To study the regenerative cell proliferation in epidermis in more detail, changes in cell cycle distributions were observed by means of micro-flow fluorometry during the first 48 hr following adhesive tape stripping. 3H-TdR uptake (LI and grain count distribution) and mitotic rate (colcemid method) were also observed. An initial accumulation of G2 cells was observed 2 hr after stripping, followed by a subsequent decrease to less than half the control level. This was followed by an increase of cells entering mitosis from an initial depression to a first peak between 5 and 9 hr which could be satisfactorily explained by the changes in the G2 pool. After an initial depression of the S phase parameters, three peaks with intervals of about 12 hr followed. The cells in these peaks could be followed as cohorts through the G2 phase and mitosis, indicating a partial synchrony of cell cycle passage, with a shortening of the mean generation time of basal cells from 83-3 hr to about 12 hr. The oscillations of the proportion of cells in G2 phase indicated a rapid passage through this cell cycle phase. The S phase duration was within the normal range but showed a moderate decrease and the G1 phase duration was decreased to a minimum. In rapidly proliferating epidermis there was a good correlation between change in the number of labelled cells and cells with S phase DNA content. This shows that micro-flow fluorometry is a rapid method for the study of cell kinetics in a perturbed cell system in vivo.     

Study of the Lineage and Cell Cycle of Small Micromeres in Embryos of the Sea Urchin, Hemicentrotus pulcherrimus

A study was made of 1st cell cycle of small micromeres, segregated at the 5th cleavage cycle, in the sea urchin embryos of Hemicentrotus pulcherrimus . For identification of small micromeres, the embryos were pulse labeled with 5-bromodeoxyuridine (BrdU) at the 1st cleavage. Using multiparametric microfluorometry equipped with a scanning stage (Tanaka, 1990), DNA content, extent of BrdU incorporation, protein content and the extent of ³H-thymidine labeling were measured on identical individual cells dissociated from an embryo. The findings of the present study are as follows. There is a short period of time between the telophase and onset of DNA replication. The period of DNA replication is 5 hr and after which, asynchronous mitosis takes place to produce 8 cells before hatching. The long S period is 83% the total 6 hr of the cell cycle. The rate of DNA accumulation is quite small during the initial one third of S but increases later in this phase. The degree of chromatin condensation remains high even during the S phase but it is low in large micromeres. The cell cycle may possibly be related causally to the development of small micromeres. The developmental significance of cell cycle duration, particularly that of DNA replication is discussed.     

Sensitivity to macrophage-mediated cytostasis is cell cycle dependent

We have examined the sensitivity of proliferating lymphoid cells in different phases of the cell cycle to macrophage-mediated cytostatic activity. These studies evaluated the ability of target cells enriched in individual cell cycle phases to pass into the next phase during brief (2–6 hr) periods of coculture with activated or nonactivated peritoneal macrophages. Both normal (concanavalin A-stimulated spleen cells) and neoplastic (Gross virus-induced thymic lymphoma) cells were analyzed. Spleen cells or lymphoma cells were first separated by centrifugal elutriation into populations highly enriched for G₁, S, or G₂/M phases of the cell cycle and cultured in the presence of nonactivated or activated macrophages for periods of 2, 4, or 6 hr. The cellular DNA content of recovered nonadherent target cells was then analyzed by flow cytometry after staining with propidium iodide. Macrophage contamination of target cell populations was insignificant under these conditions. Nonactivated macrophages did not affect target cell cycle traverse when compared with target cells cultured alone. Activated macrophage mediated cytostatic activity resulted in complete block of the transition of cells in G₁ phase into S phase and of the further accumulation of DNA by cells in early S phase. Cells already in mid to late S phase were able to continue DNA replication at rates nearly equivalent to control cells. There was no inhibition of the passage of cells through G₂ or mitosis. These effects were seen by as early as 2 hr of macrophage-target cell coculture and both normal and neoplastic cells exhibited identical patterns of cell cycle phase sensitivity.     

Studies of the mechanism by which gangliosides inhibit the proliferative response of murine splenocytes to concanavalin A

Gangliosides are known to inhibit the proliferative response of murine and human lymphocytes to antigens and mitogens in vitro. In this study the response of murine spleen cells to concanavalin A (Con A) was used as a model system. Analysis of the cellular events by flow cytometry revealed that during the first 24 hr of culture the effect of gangliosides on Con A-treated cells was minimal. At 48 hr, however, more of the ganglioside-treated cells were in G0/G1, the cells contained more RNA, and fewer cells were in S phase. These data indicate that gangliosides inhibit the transition of the cells from G0/G1 into the S phase of the cell cycle. Expression of the interleukin 2 (IL-2) receptor, as measured by the binding of a monoclonal antibody to the receptor, was not inhibited by the gangliosides. Binding of 125I-labeled recombinant IL-2 to cells cultured for 48 hr with Con A was inhibited by ganglioside GD1a but not by asialo GM1. Inhibition was much more effective if the gangliosides were preincubated with IL-2 before addition of cells, but no inhibition was observed if the cells were preincubated with gangliosides and the unbound gangliosides were washed out prior to addition of the IL-2. These data suggest that interference with the binding of IL-2 to the high-affinity IL-2 receptor of activated T lymphocytes plays an important role in the inhibition of Con A-induced proliferation.     

G1 and S phase mammalian cells synthesize histones at equivalent rates

To determine the effect of cell cycle position on protein synthesis, synchronized cell populations were metabolically labeled and the synthesis of the basic proteins, including histones, was examined by two-dimensional gel electrophoresis. Exponentially growing S49 mouse lymphoma or Chinese hamster ovary (CHO) cells were separated into G1 and S phase populations by centrifugal elutriation, selective mitotic detachment, fluorescence-activated cell sorting, or a combination of these, and pulse-labeled with radiolabeled amino acids. The histone proteins, both free and chromatin-bound, were completely resolved from some 300 other basic polypeptides in whole-cell lysates by a modification of the NEPHGE technique of O'Farrell, Goodman and O'Farrell (1977). Comparisons of matched autoradiograms from samples of G1 and S phase labeled cells revealed an equivalent rate of histone synthesis through the cell cycle of both S49 and CHO cells. Nuclei isolated from G1 phase S49 cells that were pulse-labeled contained between 13 and 15% of the newly synthesized nucleosomal histones present in S phase nuclei. Nuclei prepared from G1 phase cells that were pulse-labeled and then chased for 5 hr contained more than 90% of the labeled nucleosomal histones present in wholecell lysates. It therefore seems likely that differential alterations in the rate of histone synthesis do not occur to a significant degree as cells proceed through the cycle, but the association of newly synthesized histones with DNA takes place after the onset of DNA replication.     

Cell cycle perturbation of cultured C6 glioma cells following short-term contact with a low dose of ACNU

The purpose of this study was to investigate the cell cycle perturbation of cultured C6 rat glioma cells induced by 1-(4-amino-2-methyl-5-pyrimidyl)methyl-3-(2-chloroethyl)3-nitrosourea hydrochloride (ACNU) using simultaneous flow cytometric measurements of DNA and bromodeoxyuridine (BrdU) content. A new graphic computer program permitted the quantification of cell density in hexagonal subareas and allowed the fraction of BrdU-labeled cells with mid-S phase DNA content (FLS) to be defined in a narrow window. The cell kinetic parameters such as cell cycle time (Tc) and S phase time (Ts) were estimated from a manually plotted FLS curve at 18 and 6 hr, respectively. The major effect of ACNU on the cell cycle was an accumulation of the cells in the G2M phase 12 to 24 hr posttreatment when compared to G2M traverse of untreated cells. For the two-dimensional analysis, cells were labeled with BrdU and then treated with ACNU, or treated with ACNU and then labeled with BrdU. It was concluded that the cells in the S and G2M phases at the time of ACNU administration progressed to mitosis but that the G1 phase cells accumulated in the subsequent G2M phase. Two-dimensional FCM analysis using BrdU provided a useful tool in studying cell cycle perturbation.     

The characterization of the monoclonal antibody Th-10a, specific for a nuclear protein appearing in the S phase of the cell cycle in normal thymocytes and its unregulated expression in lymphoma cell lines

A monoclonal antibody (Th-10a) specific for the nuclear protein appearing in the S phase of the cell cycle in normal mouse thymocytes was derived by immunizing Wistar rats with a murine thymic lymphoma (TIGN), and its isotype was rat IgG_2a and had κ light chain. Immunohistochemical staining of frozen sections of B10.Thy1.1 newborn thymus and embryonic intestine revealed that this monoclonal antibody reacted strongly with the nuclear proteins of subcortical thymocytes and the basal layer of the mucosa, where many cells were dividing, but not with that of the thymic medullary area. To evaluate the expression of the nuclear proteins during the cell cycle in detail, the results of an immunofluorescence analysis of the thymocytes from hydroxyurea-treated B10 mice using Th-10a monoclonal antibody were compared with those of DNA synthesis of these cells with the use of the FITC-conjugated anti-BrdUrd monoclonal antibody. The results indicated that the nuclear protein detected by Th-10a monoclonal antibody was highly expressed in the S phase of normal thymocytes, while the cells in G₁, G₂ and M phases exhibited a low level of the expression. Moreover, the variations in expression of the nuclear proteins in the thymocytes at different times after hydroxyurea treatment were observed to correspond with the frequency of DNA synthesizing cells. In contrast, the high level and unregulated expression of the nuclear protein detected by Th-10a monoclonal antibody was observed throughout the cell cycle of the mouse lymphoma cell lines examined. Since Th-10a monoclonal antibody does not react with the nuclear proteins derived from human, hamster or rat proliferating cells, this antibody may recognize a murine specific epitope of the nuclear protein. To further characteize the nuclear proteins, we extracted them from normal thymocytes or thymic lymphomas, and analysed them by immunoblotting or***     

The cell cycle dependence of thermotolerance. II. CHO cells heated at 45.0 degrees C

This report extends our investigations of the cell cycle dependence of the expression of thermotolerance to include tolerance expressed by Chinese hamster ovary (CHO) cells exposed to 45.0 degrees C hyperthermia. We examined the response of asynchronous cells following exposure at 45.0 degrees C. A maximum in thermotolerance under these conditions was reached approximately 12 hr after a 15-min exposure to 45.0 degrees C hyperthermia and progressively decreased thereafter. Cells were delayed in S and G2 phase for 24 hr, after which time cell growth resumed. We then characterized the response of CHO cell populations synchronized in G1 or early or late S phase. We observed that the expression of tolerance depended on the position of cells in the cell cycle and was modulated by changes in the sensitivity of cells as they progressed through the cell cycle subsequent to the tolerance induction dose. We measured the variation in the sensitivity of these cells to 45.0 degrees C hyperthermia throughout the cell cycle and found substantial changes as cells progressed through S phase. Cells in early S phase were the most sensitive to heat at this temperature, and as these cells progressed through S phase, they became progressively more resistant. In addition, G1 cells were delayed for approximately 15 to 18 hr by a 15-min, 45.0 degrees C heat pulse, whereas S-phase cells were delayed to a lesser extent. The data presented in this report suggest that the induction of thermotolerance is relatively non-cell-cycle specific, but the magnitude of expression of tolerance depends on the position of cells in the cell cycle at the time of the subsequent challenge heat dose.     

REGENERATIVE PROLIFERATION OF MOUSE EPIDERMAL CELLS FOLLOWING ADHESIVE TAPE STRIPPING

The proliferating cells of mouse epidermis (basal cells) can be separated from the non-proliferating cells (differentiating cells) (Laerum, 1969) and brought into a mono-disperse suspension. This makes it possible to determine the cell cycle distributions (e.g. the relative number of cells in the G^ S and (G₂+ M) phases of the cell cycle) of the basal cell population by means of micro-flow fluorometry. To study the regenerative cell proliferation in epidermis in more detail, changes in cell cycle distributions were observed by means of micro-flow fluorometry during the first 48 hr following adhesive tape stripping. ³H-TdR uptake (LI and grain count distribution) and mitotic rate (colcemid method) were also observed. An initial accumulation of G₂ cells was observed 2 hr after stripping, followed by a subsequent decrease to less than half the control level. This was followed by an increase of cells entering mitosis from an initial depression to a first peak between 5 and 9 hr which could be satisfactorily explained by the changes in the G₂ pool. After an initial depression of the S phase parameters, three peaks with intervals of about 12 hr followed. The cells in these peaks could be followed as cohorts through the G₂ phase and mitosis, indicating a partial synchrony of cell cycle passage, with a shortening of the mean generation time of basal cells from 83-3 hr to about 12 hr. The oscillations of the proportion of cells in G₂ phase indicated a rapid passage through this cell cycle phase. The S phase duration was within the normal range but showed a moderate decrease and the Gj phase duration was decreased to a minimum. In rapidly proliferating epidermis there was a good correlation between change in the number of labelled cells and cells with S phase DNA content. This shows that micro-flow fluorometry is a rapid method for the study of cell kinetics in a perturbed cell system in vivo.     

Immunomodulations of thymocytes and splenocytes in trained rats.

The aim of this study was to describe the effects of training (running) on thymus and spleen cells in the rat. Young Wistar control rats (n = 6), rats trained for 4 wk (n = 5), and rats trained for 4 wk followed by 1 wk of intensive training (3 h/day, n = 6) were studied. Various lymphocyte surface and nuclear markers were determined by immunocytochemistry. The results show that 4 wk of training 1) decreased the percentage of bromodeoxyuridine (BrdU+) thymocytes (cell in phase S of the cycle, immature thymocytes; P less than 0.05) and the viability of thymocytes stimulated with concanavalin A (Con A; P less than 0.05) and 2) increased the absolute number of CD8+ (suppressor/cytotoxic T cells; 29%) and the percentage of CD8+ splenocytes (P less than 0.01). An additional week of intensive training in the 4-wk trained rats induced 1) a decrease in the absolute number of thymocytes (25%, P less than 0.05), TCR+ thymocytes, splenocytes (28%, P less than 0.01), T, CD4+ (helper T cells; 34%), and CD8+ (31%) splenocytes (P less than 0.01) and 2) an increase in the viability of splenocytes after stimulation with Con A for 72 h (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)