3H-thymidine Labeling Research Articles

From epidemiology to treatment: Aspirin's prevention of brain and breast-cancer and cardioprotection may associate with its metabolite gentisic acid

BackgroundEpidemiological studies indicate that aspirin consumption reduces the risk of tumors, which is especially relevant for colonic adenoma and carcinoma. Similar observations were made for glial brain tumors and breast cancers, yet the results are inconsistent. Gentisic acid (GA) is a minor catabolite of aspirin; yet humans carrying CYP2C9-variants incapable to catabolize aspirin to GA do not benefit from aspirin in prevention against colonic adenoma. GA blocks binding of Fibroblastic Growth Factor to its receptor and its sulphonate metabolite dobesilic acid blocks growth of C6 glioblastoma in vivo. GA is also an endogenously produced siderophore in mammalians for the transport of iron, a trace element which stimulates tumor growth and enhances anthracycline cardiotoxicity. Materials and MethodsIn this study, we assessed whether GA exerts direct antitumor activity on C6 glioma cells in vitro (cytotoxicity, colony growth, 3H-thymidine labeling analysis of DNA synthesis); and whether it can modify growth of Ehrlich breast ascites carcinoma (EAC) and solid tumors (EST) in vivo. GA and antitumoral trace element selenium block 12-lipoxygenase activity and aspirin's paradoxical inflammatory effects are seen in selenium-deficient humans; thus, we also investigated antitumor interactions between GA and sodium selenite. Lastly, we evaluated whether GA could protect against doxorubicin cardiotoxicity due to its function to chelate iron. ResultsClinically achievable doses of GA blocked growth, colony formation and DNA synthesis of C6 glioma in vitro with high significance. GA enhanced the survival of EAC-bearing mice at a dosage of 0.4 mg/mice/day, in which 33% of the treated animals survived more than 3-weeks, when all untreated mice succumbed to their tumors. Selenium decreased EST volumes initially, yet increased tumor volumes at later stages in surviving mice. GA alone reduced solid tumor growth and did not modify selenite antineoplasticity initially, but blocked the late tumor-stimulating effects of selenite. Lastly, doxorubicin-induced cardiac myofibrillary and endothelial damage and hyalinization necrosis were attenuated with GA treatment. ConclusionsGA highly merits to be studied in further animal models as an anticancer and chemoprotective drug.

Joseph Altman (1925–2016): A life in neurodevelopment

Joseph Altman (1925–2016): A life in neurodevelopment

Cell Proliferation of the Primary Tumor Predicts Ipsilateral Axillary Node Disease in Elderly Breast Cancer Patients

The present study investigated whether tumor markers such as cell proliferation and steroid receptor status, which have been shown to have relevance for important endpoints (relapse-free and overall survival), can also predict axillary disease in elderly patients with breast cancer. We evaluated 351 consecutive elderly women with breast cancer ≥70 years of age with estrogen receptor (ER)‑positive tumors with no palpable axillary nodes, for whom information on cell proliferation determined by the 3H-thymidine labeling index (TLI) and progesterone receptor (PgR) was available. Patients underwent quadrantectomy (70.1%) or quadrantectomy plus radiotherapy (29.9%) without axillary node dissection, followed by adjuvant tamoxifen for at least 2 years. Univariable (cumulative incidence curves) and multivariable analyses (Fine and Gray models) were carried out. After a median follow-up of 16 years, ipsilateral axillary relapse was not related to PgR status but was strongly associated with tumor cell proliferation in both small (pT1) and large (pT2-4b) tumors. Axillary relapse cumulative incidence increased from 1% in patients with low-TLI (≤3%), PgR-positive and pT1 tumors to a maximum of 20% in patients with high-TLI, PgR-negative and pT2-4b tumors. Tumor cell proliferation, determined by TLI at primary surgery, is an important predictor of axillary relapse in elderly ER-positive breast cancer patients and could help to identify patients who should undergo axillary surgery.

H3-thymidine radioautography of salivary gland chromosomes treated with alkali-urea.

The patterns of H3-thymidine labelling in the salivary gland chromosomes of Drosophila melanogaster were studied after alkali-urea treatment. Four essentially different labelling patterns were found: sparse labelling mainly or only in the central axis; spot labelling with almost continuous label in the axis and around it; labelling of whole chromosomes; spot labelling without appreciable label of the central axis. The possible chronological order for these patterns was suggested on the basis of earlier results. The labelling patterns were also interpreted according to the loop model of the lampbrush stage.

An improved method for the cell cycle synchronization of Vicia faba root meristem cells

Plant root meristem cells divide asynchronously which makes biochemical analysis of cell cycle regulation particularly difficult. In the present article a high level of cell cycle synchronization in Vicia faba root meristems was obtained by using a rich medium (HNS), special culture conditions and a double-block method with replication inhibitor—hydroxyurea (HU). Two HU concentrations were tested and different periods of the first and the second synchronization, and of cycle recommencement between the first and the second blockage. The level of synchronization was estimated on the basis of 3H-thymidine labeling indices, mitotic, and phase indices and indices determining the percentage of G1 and G2 cells, which were identified by cytophotometric measurements of DNA content in individual nuclei. The highest level of cell cycle synchronization was obtained after double treatment of meristems with 1.25 mM HU (18 and 12 h) separated by 6-h incubation in HNS without HU. During the second postincubation in HNS in subsequent hours: 4, 7, 10, 11, over 90% of cells in the S phase, nearly 70% in G2 phase, 86% in mitosis, and nearly 70% in G1 phase were received, respectively. The use of 2.5 mM HU in a similar experimental procedure caused disturbed divisions.

87 INVITED Neoadjuvant antiHER2 therapy

There is increasing evidence for a direct interaction of the enteric nervous and immune system. Receptors for neuropeptides such as VIP, somatostatin, and substance P have been characterised in human immuno-haematopoietic cells but little is known about the functional significance and expression of receptors for cholecystokinin (CCK) on cells of the immune system. There are only few studies that describe the expression of CCK receptors on human leukaemia-derived cell lines but the receptor structure and function in normal leukocytes have not been clearly established. We therefore sought to determine CCK receptor expression, structure, and function in nontransformed human peripheral blood mononuclear cells.Full-length cDNA clones encoding the human CCK-A and CCK-B/gastrin receptor are expressed in peripheral blood mononuclear cells from healthy volunteers without haematopoietic malignancy. In addition to wild-type CCK-B/gastrin receptor cDNAs, we isolated a splice variant with an in frame insertion of 69 amino acids within its putative third intracellular receptor loop. Dideoxy sequence analysis revealed that the cDNA of this splice variant comprises exons 1–4 but retains intron 4 (207 bp) in the absence of mutations within the splice donor sites. Transient expression of this splice variant in COS-7 cells reveals wild-type affinity for CCK-8, Gastrin-17, and antagonist L-365,260. Affinity for glycine-extended gastrin-17 was not increased when compared to the wild-type CCK-B/gastrin receptor. In vitro, gastrin decreased 3H-thymidine labelling in phytohaemagglutinin-pretreated mononuclear cells at a half-maximally effective concentration of 1.5 nM. We also isolated a cDNA encoding another splice variant of the CCK-B/gastrin receptor with a 158 bp deletion of the entire exon 4 sequence.We conclude that wild-type transcripts of both CCK receptor subtypes and splice variants of the CCK-B/gastrin receptor are expressed in nontransformed human mononuclear cells and that gastrin exhibits antiproliferative effects.

Modifications of nuclear envelope during differentiation and depolyploidization of rat trophoblast cells

An increased activity of membranes of the nuclear envelope (NE) was observed electron microscopically in the trophoblast cells of the rat placenta. The activity of the membranes was manifested as formation of various NE derivatives, such as the annulate lamellae (AL), the intranuclear tubules, and the concentric membranous structures. At the period of terminal differentiation of the secondary giant trophoblast cells (SGTC) the NE derivatives play active role in subdivision of the initial highly polyploid nuclei into the numerous low-ploidy fragments. 3H-thymidine labeling showed that attenuation of the DNA replication precedes the nuclear fragmentation. In the course of the nuclear fragmentation the narrow deep NE invaginations subdivide the nucleus into the separate lobes that subsequently are detached from the initial nucleus. By the beginning of the fragmentation, the accumulated membranous structures, i.e. the intranuclear AL, tubules, clusters of pore complexes, etc., seem to be the source of the reserve material that is necessary for formation of the great amount of the NE membranes of the newly formed nuclear fragments. Thus, the intranuclear membranous structures that seem to increase the active surface of the growing endopolyploid nucleus at the earlier stage of differentiation then take part in genome isolation that results in formation of a multinucleate cell with diploid and low-polyploid nuclei. The outer NE membrane of the initial nucleus plays an active role in compartmentalization of cytoplasmic areas around the nuclear fragments within the giant polykaryocyte. Apart from the membranous structures the bundles of intermediate filaments (IF) located in the cytoplasm perinuclear zones seem to participate in the nuclear fragmentation. These processes are likely to provide formation of the giant polykaryocytes incapable for further proliferation.

Heavy Water Labeling of Keratin as a Non-Invasive Biomarker of Skin Turnover In Vivo in Rodents and Humans

Measurement of skin turnover has been problematic in humans. Heavy water (2H2O) labeling has recently been developed as a safe, simple method to study in vivo kinetics of many biosynthetic processes, including DNA and protein synthesis. Here, we apply this approach to the measurement of 2H incorporation into skin keratin and show close agreement between keratin and keratinocyte turnover data in the epidermis of rodents. Elevated turnover rates of both keratin and keratinocytes were observed in the epidermis of the flaky skin mouse, although topical treatments effective in human psoriasis had no effect on either turnover rate in these mice. In humans, keratin turnover was monitored non-invasively by serial tape stripping during and after 2H2O labeling. Kinetic data were consistent with previous estimates of epidermal turnover, with a lag time of 18 days before label appeared at the skin surface and a transit time of 4-5 weeks. Variability in skin keratin turnover rates was present among healthy individuals. In summary, 2H2O labeling of skin keratin represents a non-invasive approach for assessing skin turnover dynamics in pre-clinical models and in human subjects.

Angiotensin-II activates apoptosis, proliferation and protein synthesis in the left heart ventricle of newborn albino rats

Angiotensin II (ANG-II) is a critical regulator of various signaling pathways involved in growth and remodeling of the vascular, cardiac, and renal cells and tissues. Although it contributes to several physiologic and pathologic events in the cardiovascular system, its role in growth and differentiation of the newborn heart is still unclear. We analyzed the effect of ANG-II treatment on apoptosis, DNA synthesis, and nucleolar organizer regions (AgNORs) activity in newborn rat myocardium. Injections of ANG-II for 5-days caused significant increase of the 3H-thymidine labeling index ( M ± m) in the myocardium of 7-day-old rats (from 6.95 ± 0.32% to 8.53 ± 0.22%, p < 0.05). There was also significant increase in the cross sectional surface area of cardiomyocytes (from 686 ± 57 to 872 ± 54 μm 2, p < 0.05), number of nucleoli (from 2.5 ± 0.05 to 2.8 ± 0.1, p < 0.05), and nucleolar surface area (from 2.6 ± 0.09 to 3.2 ± 0.22 μm 2, p < 0.05). These changes were accompanied by significant increase in the apoptotic indices analyzed by TDT-mediated dUTP-biotin nick end-labeling (TUNEL) (from 0.044 ± 0.01% to 0.093 ± 0.01%, p < 0.05). Interestingly, we found no differences in cell proliferation between the test and control animals after 21–45 days of age, which were injected with ANG-II in the first postnatal week. However, the area of cardiomyocytes and the number of nucleoli in 21-day-old rats continued to increase significantly. Our results indicate that ANG-II modulates cardiac growth during the neonatal period via stimulation of apoptosis, cell cycle events and cellular growth of cardiomyocytes and that these effects can persist up to 15 days after injection of ANG-II has been completed.

G1 Phase Regulation, Area-Specific Cell Cycle Control, and Cytoarchitectonics in the Primate Cortex

We have investigated the cell cycle-related mechanisms that lead to the emergence of primate areas 17 and 18. These areas are characterized by striking differences in cytoarchitectonics and neuron number. We show in vivo that (1) area 17 precursors of supragranular neurons exhibit a shorter cell cycle duration, a reduced G1 phase, and a higher rate of cell cycle reentry than area 18 precursors; (2) area 17 and area 18 precursors show contrasting and specific levels of expression of cyclin E (high in area 17, low in area 18) and p27Kip1 (low in area 17, high in area 18); (3) ex vivo up- and downmodulation of cyclin E and p27Kip1 show that both regulators influence cell cycle kinetics by modifying rates of cell cycle progression and cell cycle reentry; (4) modeling the areal differences in cell cycle parameters suggests that they contribute to areal differences in numbers of precursors and neuron production.

Cytoprotective effects of amifostine and cysteamine on cultured normal and tumor cells treated with paclitaxel in terms of mitotic index and 3H-thymidine labeling index

In this study, the cytotoxic effects of paclitaxel (PAC) in normal and tumor cells were established, and the cytoprotective effects of amifostine (AMI) and cysteamine (CYS) against this cytotoxicity were examined. Tumor cell lines used in this study were L-strain cells of mouse subcutaneous origin and human cervix carcinoma-derived HeLa cells. Mouse embryonic fibroblasts (MEFs) were used as the normal cell line. Results of the experiments were evaluated in terms of the mitotic index and the 3H-thymidine labeling index. PAC concentrations of 6 and 12 microg/ml were applied to the cells for 1-10 days either alone or in combination with 1 mug/ml of AMI and CYS. In terms of the above parameters, statistically significant effects were not seen in cultures of any of the cell lines treated with 1 microg/ml of AMI or CYS alone. In contrast, both concentrations of PAC caused increasing cytotoxic effects with increasing treatment time (P < .001). The cytotoxic effect of PAC appeared as mitotic phase accumulation (G2/M blockage) and a subsequent decline in the synthesis phase. HeLa cells were very sensitive to PAC treatment, whereas MEF cells were quite resistant compared with tumor cells. In cells treated with combined drugs to investigate the cytoprotective effects of AMI and CYS on normal and tumor cell lines, PAC continued to show cytotoxic effects in tumor cells, but this effect was reduced in the normal cells. AMI and CYS did not protect tumor cells against the cytotoxic effects of PAC, but protection was observed in normal cells. Furthermore, the protection provided by AMI was stronger than that provided by CYS.

Protein Phosphorylation During the Transitions from G1 Arrest Point to S and G2 Arrest Point to M in Vicia faba Root Meristem

Using monoclonal FITC conjugated antibodies that specifically recognize phosphorylated form of threonine (α-TP-FITC) it was shown that in excised, sugar-starved root meristems of Vicia faba subsp. minor the expression of two principal control points (PCPs) is correlated with a marked decrease in nuclear and nucleolar phosphorylation of proteins. When stationary phase meristems are supplied with 2 % sucrose, the G1-arrested cells start out DNA replication (assessed by pulse 3H-thymidine labeling), while the cells blocked in G2 phase initiate mitotic divisions. Using this model, we have found that depending on the period during which the sucrose-mediated PCP1→S and PCP2→M transitions are affected by 6-dimethylaminopurine, an inhibitor of protein kinases, the release from G1 and G2 phase arrest-points of the cell cycle become prolonged, showing different time-course modifications. Furthermore, our results seem to implicate that both PCP1 and PCP2 comprise subpopulations of cells differing with respect to the rates at which they commence DNA replication and mitosis.

Thymidine labeling index: prognostic role in breast cancer.

The aim of this study is to evaluate the prognostic role of thymidine labeling index in patients with breast cancer. Cellular proliferation rates in 155 breast cancer specimens were investigated by 3H-thymidine labeling index (3H-TLI). Median age was 47 years (range: 23-76). At presentation, 11 patients (7.1%) had stage I disease, 76 (49%) had stage II, 64 (41.3%) had stage III disease, and 4 (2.6%) had metastatic involvement. Patients were placed in 2 groups based on their proliferative indices. The cut-off level was assigned as the median TLI value of the whole group. Correlations between proliferative activity of the tumors based on 3H-TLI levels and various previously established prognostic factors, as well as the influence of proliferative activity on survival as a clinical outcome, were analyzed. The mean and median TLI values for the whole group of patients were 4.36 +/- 4.96% and 2.76% (range: 0-23.6), respectively. There was a significant association of nuclear grade with TLI (P = 0.04). Patients who were alive with no sign of disease at the final follow-up examination had a significantly lower median TLI rate than those who were either alive with disease or those who had eventually died with disease progression (3.7% versus 1.9%, respectively; P = 0.04). Patients with locally advanced disease (N2 + N3 involvement) had a significantly higher median TLI rate than those with local nodal involvement (N1) (3.4% versus 1.7%, respectively, P = 0.026). Furthermore, TLI levels showed a significant association with overall survival in patients with node-negative disease (P = 0.02). Based on the results of this study, it can be concluded that TLI plays a significant prognostic role in a subset of patients with node-negative breast cancer. Furthermore, TLI appears to have a predictive value for the clinical outcome of patients with breast cancer. These findings may justify a more aggressive therapeutic approach in patients with high TLI levels. Further large-scale, prospective studies are required before a definite conclusion can be reached.reached.

Peritoneal fluid-mediated enhancement of eutopic and ectopic endometrial cell proliferation is dependent on tumor necrosis factor-α in women with endometriosis

Objective: To determine the effect of autologous peritoneal fluid and tumor necrosis factor-α (TNF-α) on proliferation of endometrial cells from women with endometriosis. Design: Endometrial cells from eutopic and ectopic endometrium were cultured in vitro with peritoneal fluids or recombinant TNF-α for 72 hours before DNa synthesis determination by 3H-thymidine labeling and liquid scintillation counting. Setting: An institute for the study and treatment of endometriosis and university-based research laboratories. Patient(s): Thirty-five women with endometriosis and 17 controls without endometriosis. Main Outcome Measure(s): In vitro incorporation of 3H-thymidine in endometrial cells was examined. Result(s): Peritoneal fluid from women with endometriosis enhanced proliferation of autologous and heterologous endometrial cell cultures from women with endometriosis. The soluble TNF-receptor etanercept blocked the ability of peritoneal fluid from women with endometriosis to enhance proliferation of eutopic or ectopic endometrial cells. Recombinant TNF-α also enhanced proliferation of eutopic and ectopic endometrial cells from women with endometriosis. In contrast, autologous peritoneal fluid, heterologous peritoneal fluid from women with endometriosis, and recombinant TNF-α failed to enhance, and often inhibited, the proliferation of eutopic endometrial cells from controls without endometriosis. Conclusion(s): Endometrial cells from women with endometriosis can utilize factors in peritoneal fluids, such as TNF-α, to facilitate proliferation in ectopic environments. Endometrial cells from women without endometriosis do not share this ability, suggesting that this abnormality is etiologically related to development of the disease. Therapy with agents that block the effects of TNF-α may be warranted.

Staurosporine and vanadate can induce additional endo-S phases during cell differentiation in primary roots of Pisum sativum

Using Feulgen-DNA cytophotometry and 3H-thymidine labeling, the effects of staurosporine (ST) (a potent inhibitor of cyclin-dependent kinases, CDKs) and sodium vanadate (NaVO 3, an inhibitor of protein phosphatases) were investigated on DNA endoreduplication during cell differentiation in the primary roots of Pisum sativum. The obtained results indicate that cellular commitment to endo-S phase may appear as either (1) a consequence of suppression imposed directly on CDK activity or (2) an indirect output connected with the decreased activity of protein phosphatases, including putative Cdc25-like enzyme indispensable to turn the switch on for appropriate conformation of the CDK/B-type cyclin complex. Furthermore, the effects induced by the combined application of ST and vanadate have been found considerably greater than the outcome produced by each of the inhibitors applied separately. Despite evident synergy, root cell nuclei stimulated by ST/vanadate mixture show significantly decreased intensity of 3H-thymidine incorporation. It is concluded that opposite influences allow a greater number of cells to commence DNA endoreduplication, but extend the times needed to traverse the extra rounds of DNA synthesis.

Identification of CCK-B/gastrin receptor splice variants in human peripheral blood mononuclear cells

There is increasing evidence for a direct interaction of the enteric nervous and immune system. Receptors for neuropeptides such as VIP, somatostatin, and substance P have been characterised in human immuno-haematopoietic cells but little is known about the functional significance and expression of receptors for cholecystokinin (CCK) on cells of the immune system. There are only few studies that describe the expression of CCK receptors on human leukaemia-derived cell lines but the receptor structure and function in normal leukocytes have not been clearly established. We therefore sought to determine CCK receptor expression, structure, and function in nontransformed human peripheral blood mononuclear cells. Full-length cDNA clones encoding the human CCK-A and CCK-B/gastrin receptor are expressed in peripheral blood mononuclear cells from healthy volunteers without haematopoietic malignancy. In addition to wild-type CCK-B/gastrin receptor cDNAs, we isolated a splice variant with an in frame insertion of 69 amino acids within its putative third intracellular receptor loop. Dideoxy sequence analysis revealed that the cDNA of this splice variant comprises exons 1–4 but retains intron 4 (207 bp) in the absence of mutations within the splice donor sites. Transient expression of this splice variant in COS-7 cells reveals wild-type affinity for CCK-8, Gastrin-17, and antagonist L-365,260. Affinity for glycine-extended gastrin-17 was not increased when compared to the wild-type CCK-B/gastrin receptor. In vitro, gastrin decreased 3H-thymidine labelling in phytohaemagglutinin-pretreated mononuclear cells at a half-maximally effective concentration of 1.5 nM. We also isolated a cDNA encoding another splice variant of the CCK-B/gastrin receptor with a 158 bp deletion of the entire exon 4 sequence. We conclude that wild-type transcripts of both CCK receptor subtypes and splice variants of the CCK-B/gastrin receptor are expressed in nontransformed human mononuclear cells and that gastrin exhibits antiproliferative effects.

Biomarker prediction of clinical outcome in operable breast cancer patients treated with tamoxifen.

The predictivity of tumour size, oestrogen (ER) and progesterone (PgR) receptors, 3H-thymidine labelling index (TLI), c-erbB-2 and p27kip1 expression on clinical outcome was analysed on a consecutive series of 118 postmenopausal patients with ER-positive, node-positive tumours. All patients were treated with surgery +/- radiotherapy and adjuvant tamoxifen (30 mg/day) for at least 2 years. TLI, ER, c-erbB-2 and p27kip1 were generally unrelated to each other. PgR was directly related to ER and inversely to c-erbB-2. Tumour size was inversely related to both c-erbB-2 and p27kip1 expression. At a median follow-up of 75 months, 5-year relapse-free survival was significantly lower for patients with very rapidly proliferating (HR = 2.61, 95% CI = 1.34-5.08), PgR negative (HR = 2.76, 95% CI = 1.43-5.33) or relatively low ER content (HR = 2.20, 95% CI = 1.14-4.25) tumours than for patients with tumours expressing the opposite biological profiles. Overall survival was also significantly different as a function of TLI (HR = 3.47, 95% CI = 1.52-7.93) and PgR (HR = 2.27, 95% CI = 1.00-5.15). TLI and PgR maintained an independent relevance in multivariate analysis and together were capable of identifying subgroups of patients at significantly different risk of relapse and death.

Predictability of monthly and yearly rhythms of breast cancer features.

In order to evaluate if breast cancer biological characteristics undergo significant menstrual and seasonal variations, we analysed in a consecutive series of 905 breast cancer patients, steroid receptor level (ER and PgR by DCC assay), proliferative activity (3H-Thymidine Labeling Index, 3H-TLI) and size of primary tumour in relation to calendar date and day of menstrual cycle at the time of the surgical procedure. For data analysis, the method of time series construction and classical spectral analyses with Bartlett Kolmogorov-Smirnov test for white noise (BKS test) was utilised. For what concerns menstrual variations, 3H-TLI showed a significant periodicity (t = 0.3146, p < 0.01 by BKS test) with peaks at day 12nd and day 18th; ER showed a significant periodicity (t = 0.3605, p < 0.01 by BKS test) with more evident peak at day 27th; PgR, a significant periodicity (t = 0.160, p = 0.05 by BKS test) with peaks at day 15th and day 24th, similar to that observed for tumour size (t = 0.19, p < 0.05 by BKS test). With respect to yearly fluctuations, 3H-TLI showed only a trend for a significant rhythm (t = 0.16, p = 0.06 by BKS test) with peaks in May and November; ER a significant periodicity (t = 0.2099, p < 0.05 by BKS test) with two evident peaks in January and April; also for PgR a significant periodicity (t = 0.3161, p < 0.05 by BKS test) was demonstrated with a peak in July; finally, tumour size showed a significant rhythm (t = 0.335, p < 0.01) paralleling 3H-TLI behaviour. Finally, the analysis of variance with interaction of menstrual and seasonal timings showed that only the seasonal timing was able to independently influence the 3H-TLI variations (3H-TLI higher in spring). We confirmed that breast biology has significant menstrual and seasonal variations and that the seasonality is probably the timing factor more relevant in periodicity determination.

Effect of cytomegalovirus on cell cycle progression and formation of pathological mitoses in cultured human diploid fibroblasts

The effect of cytomegalovirus on the cell cycle was studied autoradiographically in an asynchronous culture of human diploid fibroblasts. The analysis of labeled mitosis showed that some cells infected in the S phase ceased to progress through the cell cycle at one of its phases (S, G2, or M); at the same time, at least part of infected cells remained capable of entering mitosis. Beginning from day 2 after infection by cytomegalovirus, the accumulation of pathological mitotic cells blocked at metaphase was observed in the culture. Approximately 50% of these cells contained 3H-thymidine label above chromosomes. This fact suggested the possibility of pathological mitosis in cells that were infected both at the S and other phases of the cell cycle. The detailed morphological analysis of chromosomes at different stages of infection demonstrated that the degree of their morphological changes increases from slight (stronger condensation) to severe pathology (fragmentation). In the aggregate, the results of the study suggested that abnormal chromosome morphology resulted from irreversible cell division arrest under the effect of cytomegalovirus.

Labeling DNA with stable isotopes: economical and practical considerations.

Labeling DNA with stable isotopes to measure cell proliferation can be a technique as effective as 3H-thymidine labeling without the limitations imposed by using radioisotopes. Here, we investigated the relative efficiency of four nonradioactive precursors to DNA: [1-13C]-glycine, [1,2-13C2]-glycine, [U-13C]-glucose, and [U-13C, 15N]-thymidine. The efficiency of incorporation for each of these labeled precursors in HEP G2 cells in culture has been studied. When considering the actual costs of in vivo experiments in which large doses of labeled material are needed, economical constraints may play an important role in defining a practical method. Therefore, the economics of this process were also considered. Using the enrichment per dollar for whichever nucleoside had the highest incorporation in a given experiment, glycine is about five times more economical as a label than thymidine and eight times more economical than glucose in these cells.