bluuebunny/crossref_metadata_2025 huggingface dataset:
The G1 interval in the mammalian cell cycle: dual control by mass accumulation and stage‐specific activities
Authors: Murray L. E. , Singer R. A. , Fenwick R. G. , Johnston G. C.
Published: May 2008

Abstract. The temporal determinants of the G1 cell cycle interval were investigated using nine mammalian cell lines. In each case, cells were allowed to proliferate for many cell cycles under conditions that slowed progress through S phase without an equivalent impairment of overall mass accumulation. This disproportionate inhibition of progress through the cell cycle caused newly produced cells to be more massive than usual. Under these growth conditions, the determinants of the length of the G1 interval became evident.

For two cell lines, HeLa S3 and NIH 3T3, a protracted S phase, and the resultant increase in mass, resulted in a dramatically shortened G1 interval. Thus, for these cell lines, a major portion of G1 time exists to accommodate mass accumulation needed to initiate the subsequent S phase. Nevertheless, under conditions that protracted S phase and shortened the G1 interval, cells still exhibited a measurable G1 time, reflecting the stage‐specific activities within G1. One activity that may be responsible for this obligatory G1 time is the synthesis of a labile protein.

For other cells studied here, protraction of S phase also caused proliferating cells to become more massive, but in these cases there was no diminution of the G1 time. For these cells, the entire G1 interval must accommodate G1‐specific activities necessary to initiate a new cell cycle.

A unifying view of the G1 interval recognizes the two distinct influences that determine the time spent in G1: the need to accumulate sufficient mass to initiate a new DNA‐division sequence; and the stage‐specific events necessary for the subsequent S phase. The length of the G1 interval is dictated by the longer of these two time‐consuming activities.
The effect of chemical radiation protectors on cell cycle progression after gamma or neutron irradiation
Authors: Sigdestad C. P. , Bergquist B. L. , Grdina D. J.
Published: May 2008

Abstract. The effects of two chemical radiation protectors, WR‐1065 and WR‐151326, were characterized in V79 Chinese hamster cells after either cobalt‐60 (60Co) gamma or fission spectrum neutron irradiation. Each protector was administered at a concentration of 4 mM to exponentially growing cultures for 30 min prior to and during irradiation with either 60Co gamma or JANUS fission spectrum neutrons. After irradiation the cells were either plated immediately for survival or returned to the incubator and assayed for cell progression. Aliquots of cells were removed at selected times, counted, fixed and stained with 4'6‐diamidino‐2‐phenylindole (DAPI). Analysis of DNA histograms indicate that the presence of the protector during irradiation reduced the division delay experienced at the G2‐M interface. Implications of these effects are discussed.
Characterization and cell cycle kinetics of hepatocytes during rat liver regeneration: in vivo BrdUrd incorporation analysed by flow cytometry and electron microscopy
Authors: Vitale M. , Rizzoli R. , Mazzotti G. , Zamait L. , Galanzi A. , Rizzi E. , Manzoli L. , Matteucci A. , Papa S.
Published: May 2008

Abstract. The incorporation of bromodeoxyuridine (BrdUrd) into newly synthesized DNA has been analysed during hepatocellular regeneration induced by partial hepatectomy in young rats. The kinetic state of the liver has been studied by flow cytometric analysis of the incorporated BrdUrd, while the fine localization of DNA replication sites through the cell cycle has been investigated at the ultrastructural level by the immunogold technique. Eighteen hours after partial hepatectomy flow cytometry revealed an early S phase distribution which corresponded to a specific staining of the interchromatin domains of the hepatocyte nucleus. Thirty‐four hours after hepatectomy, on the other hand, when most cells were in late S, a specific staining of heterochromatin domains was observed. The effect of the BrdUrd technique on nuclear aggregation has also been analysed and discussed. The results demonstrate that specific patterns of DNA replication can be recognized during the cell cycle and that flow cytometry and electron microscopy appear to be complementary in the kinetic study of liver regeneration.
Age‐related changes in the cell proliferation of ATPC+ mouse ascites tumour
Authors: Viola‐Magni M. P. , Biagetti M.
Published: May 2008

Abstract. The growth of ATPC+, an ascites tumour derived from a spontaneous mammary carcinoma in BALB/c+ mice, was studied at different ages. It was observed that the number of cells increases rapidly during the first 5 days after implantation. Thereafter, the cell number increases more slowly, reaching a plateau after 8 days. This slowing‐down is not due to a reduction in the growth fraction but to a lengthening of the cell cycle. Between 5 and 8 days the duration of all phases increases, including the S phase, which increases from 5·2 h in 5‐day tumours to 8·2 h in 8‐day tumours. In 12‐day tumours both the cell cycle and S phase are only slightly longer than in 8‐day tumours whereas the growth fraction is reduced. The slowing‐down of cell growth can be attributed to growth fraction reduction rather than cell loss, which is maximal in the 5‐day tumour. At this age the time course of the percent labelled cells and of the number of grains/nucleus suggests reutilization of [3H]‐thymidine. Incorporation of [3H]‐thymidine/cell decreases sharply in 12‐day tumours due to a reduced availability of thymidine, which is degraded to thymine in the in vivo ascitic fluid faster than in 8‐day tumours. This indicates an age‐related change in the ascitic fluid composition.
Changes in cell cycle and chromatin distribution in C6 glioma cells treated by dibutyryl cyclic AMP
Authors: Mareš V. , Giordano P. A. , Pellicciari C. , Scherini E. , Lisà V. , Bottone M. G. , Bottiroli G.
Published: May 2008

Abstract. C6 glioma cells in culture were treated with 1 mm dibutyryl cyclic AMP (Db‐cAMP) for 5,8,24 and 72 h. The cells were labelled with [3H]‐thymidine before either the end, or the beginning, of the Db‐cAMP treatment. The cell cycle passage was monitored by the simultaneous determination of DNA content and DNA synthesis in propidium iodide stained autoradiograms. The data revealed an early (t ≤ 3–8 h) and moderate inhibitory effect of Db‐cAMP on all phases of the cell cycle except mitosis; some cells (2%) were completely blocked in the S phase. Later (8 < t < 24–72 h), the cycling of a substantial part of the population became inhibited in G1 phase. Microdensitometric texture analysis of Feulgen‐stained nuclei, performed 24 h after administration of Db‐cAMP, showed a higher inhomogeneity of the DNA distribution in cell nuclei, caused by the condensation of a part of the chromatin. This may reflect either changes in genome expression taking part in the process of cAMP induced differentiation or transit of some cells into quiescent G0 or S0 phases.
Cell cycle related studies on thymidine kinase and its isoenzymes in Ehrlich ascites tumours
Authors: He Q. , Skog S. , Tribukait B.
Published: May 2008

Abstract. Thymidine kinase (TK) activity was measured in relation to the cell cycle of in vivo growing ascites tumour cells. The cells were synchronized by means of centrifugal eiutriation and the cell cycle composition of the cell fractions was determined by flow cytometry. TK activity was low in GI, increased during S phase and declined in G2. A half‐life of TK activity of about 45 min was found throughout the cell cycle.

Four isoenzymes at PI values of 4.1, 5.3, 6.9 and 8.3, denoted as isoenzymes 1–4, were identified using isoelectric focusing. Isoenzymes 3 and 4 were responsible for the profound cell cycle related changes in the TK activity. Corresponding isoenzymes were also found in the fetal mouse liver. In the adult mouse liver isoenzyme 2 was the dominating isoenzyme. The half‐life of the isoenzymes was in the same range as for the total TK activity.

We conclude that the low TK activity in G1 is due to degradation of the enzyme in G2 at a normal rate combined with an arrest in the synthesis of TK. We also conclude that isoenzyme 4 and the intermediate isoenzyme 3, which had earlier been suggested to be a mitochondrial form of TK, in fact represent cytoplasmatic forms of TK. According to cell cycle and PI studies, isoenzyme 2 belongs to the mitochondrial form. Studies with various phosphor donors and specific substrates, however, indicate that it also contains a cytoplasmic component.
The serum response element and an AP‐1/ATF sequence immediately downstream co‐operate in the regulation of c‐fos transcription
Authors: Morgan I. M. , Birnie G. D.
Published: May 2008

Abstract. Transcription of the c‐fos gene is activated in response to a wide variety of extracellular stimuli and several cis‐acting transcriptional control elements have been characterized. One of these elements is called the serum response element (SRE) and here we investigate an interaction between this element and an AP‐1/ATF‐like sequence immediately downstream from the SRE. In growing cells these sequences activate transcription in an additive fashion whereas in quiescent cells they co‐operate to repress transcription. This co‐operation is disrupted upon separation of the elements which also alters the response of the elements to serum or 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA) stimulation of quiescent cells. This separation also results in an increase of transcription in growing cells. A consensus AP‐1 DNA‐binding site can substitute for the AP‐1/ATF‐like sequence present in the c‐fos promoter to acti transcription in an additive fashion with the SRE in growing cells, and co‐operate in repression in quiescent cells. These observations show that any interaction that may be occurring between proteins binding to these elements results in a different pattern of transcriptional control in growing and quiescent cells. Alternatively, different proteins (or modified proteins) may complex with these sequences in the two different states of cell growth.
Growth fraction measured using the comet assay
Authors: Olive P. L. , Banáth J. P.
Published: May 2008

Abstract. Growth fraction, an important determinant of tumour response to therapy, was measured using a novel assay in WiDr human colon carcinoma cells grown as monolayers, spheroids, or xenografts. The assay is based on the fact that the anti‐cancer agent etoposide produces DNA strand breaks in proliferating but not non‐proliferating cells. Strand breaks were detected in individual cells using the alkaline ‘comet’ assay, and growth fraction was defined as the fraction of cells containing elevated numbers of DNA strand breaks. The specificity of the method for detecting proliferating cells was verified directly by allowing cells to incorporate bromo‐deoxyuridine (BrdUrd) into DNA, followed by exposure to etoposide and treatment of the comets with anti‐BrdUrd antibodies. All cells stained with anti‐BrdUrd antibodies were also damaged by etoposide. Similarly, growth fraction measured using Ki‐67 correlated well with the new assay. The accuracy, speed and convenience of the comet assay for measuring growth fraction suggest that it may be useful for predicting response of human cancers to therapy.
cAMP effect on extracellular matrix synthesis in human breast cancer cells
Authors: Revillion F. , Vandewalle B. , Lassalle B. , Lefebvre J.
Published: May 2008

Abstract. The effect of a cAMP derivative (N6, 02‐dibutyryl cyclic adenosine 3′3′‐monophosphate: dBcAMP) on the cell cycle and on the synthesis of typical extracellular matrix (ECM) components, i.e. collagen and glycosaminoglycans (GAG), was studied in two hormone‐responsive human breast cancer cell lines VHB‐1 and MCF‐7. The data showed that dBcAMP induced a decrease in the proportion of cells in S + G2+ M phases due to an increase of the non‐cycling (Go phase) cell number as revealed by the Ki‐67 antigen immunocytochemical study. The collagen synthesis, estimated by [3H] proline incorporation into the cellular proteins followed by an enzymatic digestion with highly purified bacterial collagenase, was not modified by dBcAMP. In contrast, the GAG synthesis, analysed by [3H] glucosamine incorporation, was increased two‐fold in the dBcAMP treated cells. As a comparison we also tested 4‐hydroxy‐Tamoxifen (4‐OH‐Tam) since it induces similar cell cycle perturbations as dBcAMP. However, we did not observe a stimulation of the GAG synthesis following 4‐OH‐Tam treatment. These data demonstrated that the increased GAG synthesis is due to cAMP and is not a consequence of perturbations in the cell cycle. We can therefore assume that the ECM modifications induced by dBcAMP may contribute to the growth inhibition of the hormone‐responsive human breast cancer cells.
Comparison and characterization of retinal pericytes and retinal pigment epithelial cells on subcellular IP3‐sensitive Ca2+ pools
Authors: Li W. , Ye X. , Tao L. , Zhan Y. , Cohen S. , Yanoff M.
Published: May 2008

Abstract. A comparative study of inositol 1,4,5‐trisphosphate (IP3)‐induced Ca2+ mobilization in bovine retinal capillary pericytes (BRCP) and bovine retinal pigment epithelial cells (BRPE) was carried out. Both cells were permeabilized with saponin. The two cell types had similar basal levels of [Ca2+]i (130 nM for BRCP, 132 nM for BRPE) and responded to IP, in a dose‐dependent manner. However, when stimulated by various concentrations of IP3 (1–10 μM), the increase in [Ca2+]i of BRCP was always two‐ to threefold higher than that in BRPE. Subcellular‐fractionation studies showed that a single population of IP3 binding site with a high affinity and high specificity of IP3 mainly localized to plasma membrane in these two cell types. Although the dissociation constant of specific [32P]‐IP3 binding sites (Kd 1.9–2.8 nM) was similar, the profile of maximal binding capacity (Bmax) of each fraction was markedly different. In comparison, plasma membrane fractions of BRCP were with Bmax of 165 fmol/mg protein versus 90 fmol/mg protein for BRPE membranes. The ATP‐dependent Ca2uptake and IP3‐dependent Ca2+ release were observed in the both plasma membrane fractions. With quantitative correlation, the membrane fraction (2 mg) of BRCP released 0.2 nmol Ca2+ whereas BRPE only released 0.07 nmol Ca2+ with the same dose of IP3 (5 μM). The selectively higher density of IP, binding sites in coupling to the larger Ca2+‐release in the membrane of BRCP suggests that the quantity of Ca2+ mobilized is determined by the spatially preferential distribution of membrane‐associated IP3 binding sites. These findings may provide an explanation for the differences observed between BRCP and BRPE in IP,‐induced DNA replication.
Application of a DNA double labelling method for the flow cytometric analysis of recruitment of non‐cycling cells in a mixed population of P and Q cells
Authors: Bakker P. J. M. , De Vries R. J. L. , Tukker C. J. , Hoebe R. A. , Barendsen G. W.
Published: May 2008

Abstract. In this paper we describe the application of a non‐radioactive DNA double labelling and staining method to an analysis of cell proliferation kinetics by flow cytometry, aimed at the direct measurement of recruitment rates in cell cultures. The method is based on the application of two halogenated deoxyuridines: iododeoxyuri‐dine (IdUrd) and chlorodeoxyuridine (CldUrd) which are incorporated into DNA synthesizing cells. By applying two commercially available monoclonal antibodies both deoxyuridines can be detected separately. To measure recruitment all proliferating cells in a plateau phase culture were labelled first with IdUrd applied during a time interval approximately equal to the cell cycle time. Subsequently, recruitment induced by a medium change was analysed by flow cytometric assessment of incorporation of CldUrd in cells which had not taken up IdUrd.

Experiments designed to determine the toxicity of continuous labelling with IdUrd in different concentrations and of pulse labelling with CldUrd showed that there was no effect on the progression of cells through the cell cycle. The aim of this study is to test the sensitivity of the procedure to detect changes in proliferation kinetics, in particular the entrance of resting cells into the S phase. Although the cell culture model used is very simple, the results demonstrate clearly that a low rate of recruitment can be detected. It is suggested that the procedure described here is specific and sensitive enough to quantify changes in cell proliferation in tumours induced by various treatments and has advantages over other methods, which measure recruitment indirectly, or directly by using two radioactive thymidines.
The role of the IGF‐1 receptor in the stimulation of cells by short pulses of growth factors
Authors: Yoshinouchi M. , Baserga R.
Published: May 2008

Abstract. Balb/c 3T3 cells require prolonged stimulation by serum or growth factors to enter DNA synthesis. However, in p6 cells, a derivative cell line from 3T3 cells which constitutively over‐express the insulin‐like growth factor‐1 (IGF‐1) receptor, a serum pulse of only 1 h is sufficient for maximal stimulation. Furthermore, maximal stimulation of DNA synthesis is also obtained when 3T3 cells, serum‐stimulated for only 1 h, are subsequently incubated with IGF‐1. Our results indicate that short pulses of growth factors render 3T3 cells capable of responding to IGF‐1, either by increasing the number of IGF‐1 receptors or by providing a new substrate for the activated receptor.
Mitotic CDC2 kinase is negatively regulated by cAMP‐dependent protein kinase in mouse fibroblast cell free extracts
Authors: Hohmann P. , DenHaese G. , Greene R. S.
Published: May 2008

Abstract. CDC2 kinase activity was decreased by up to 75% when mitotic cell free extracts from mouse fibroblasts were incubated with cAMP and ATP. This effect was blocked by PKI, the heat stable inhibitor of cAMP‐dependent protein kinase (PKA). An acidic, heat stable protein from G1 cells, consistent with inhibitor‐1 of protein phosphatase 1, mimicked the effect of cAMP, but was not antagonized by PKI. Okadaic acid, another inhibitor of protein phosphatase 1, also downregulated CD2 activity, and the effect was independent of both cAMP and PKI. The evidence suggests that PKA exerts its effect by activating inhibitor‐1 by phosphorylation, and that the next step in the regulatory pathway requires the inactivation of one or more protein phosphatase 1 isoenzymes. Non‐denaturing gel electrophoresis suggested that the size and/or charge density of the CDC2 kinase complex was changed when the activity was downregulated by cAMP or G1 extracts.
Persistence of cell cycle times over many generations as determined by heritability of colony sizes of ras oncogene‐transformed and non‐transformed cells
Authors: Axelrod D.E. , Gusev Y. , Kuczek T.
Published: May 2008

Abstract. The persistence of cell lifetimes during about 10 successive cell generations was investigated by comparing the number of cells in primary colonies and in secondary colonies derived from primary colonies. Primary colonies were grown from single cells for 3 or 4 days (a time equivalent to an average of five cell generations) and the number of cells in each primary colony determined. Cells in each primary colony were dispersed to initiate secondary colonies, grown for the same time, and the number of cells in secondary colonies determined. Several criteria were used to compare primary and related secondary colonies, the most informative was found to be regression and correlation coefficients between number of cells in primary colonies and mean numbers of cells in related secondary colonies. For two non‐transformed mouse fibroblast cell lines, NIH 3T3 and BALB 3T3, the regression and correlation coefficients of cell number in primary and secondary colonies were positive. This suggests inheritance of cell lifetimes over many cell generations. After the addition of an activated ras oncogene (human cellular Harvey ras, or viral Kirsten ras) some regression and correlation coefficients changed in magnitude but all remained positive. Comparison of experimental data and the results of computer simulations suggest that several models of inheritance of cell lifetimes are not adequate to explain the results, including a model of independence between lifetimes of mother and daughter cells and the common model that describes daughter cells as inheriting the lifetime of their mother with deviation. Simulations do suggest that cell lifetimes are inherited within clones as deviation from the lifetime of the initial cell, and that the ras oncogene does not destroy persistence within clones but does increase heterogeneity of cell lifetimes.
Characterization and enumeration of spontaneously proliferating human leucocytes by multiparameter flow cytometry
Authors: Johannisson A. , Amnéus H.
Published: May 2008

Spontaneously proliferating human leucocytes have been characterized and enumerated using multiparameter flow cytometry. The frequency of spontaneously proliferating cells amongst human peripheral blood mononuclear cells, was determined on the basis of BrdUrd incorporation and total DNA content in samples of cells incubated in medium without added mitogen for several days. The frequency of proliferating cells decreased from an initial level of ≈ 6 × 10–4 to 3 × 10–4 after 30 h of incubation, and then rose to ≈ 2 × 10–2 after 100 h of incubation. In one reference person, the frequency showed only minor variation in this pattern over a 1.5 year interval. Simultaneous measurement of proliferation and determination of immuno‐logical subclass, as indicated with Hoechst 33342 staining and surface markers, showed an over‐representation of CD19‐positive cells, compared with CD2‐positive cells and subsets of CD2‐positive cells (CD4‐positives and CD8‐positives). This method can be used as an indicator of exposure to agents when results from animal tests are to be compared with results from human populations. The advantages are that no cell culturing is needed to perform the test, it provides the possibility of further characterizing proliferating cells, and the rapid flow‐cytometric enumeration.
The role of the IGF‐I receptor in the growth and transformation of mammalian cells
Authors: Baserga R. , Sell C. , Porcu P. , Rubini M.
Published: May 2008

Abstract. Recent developments in the molecular biology of the insulin‐like growth factor I (IGF‐I) receptor have clarified its role in cellular growth and transformation. Although cells homozygous for a targeted disruption of the IGF‐I receptor genes can grow in serum‐supplemented medium, the IGF‐I receptor is required for optimal growth, and is required equally in all phases of the cell cycle. The receptor plays an even more stringent role in cellular transformation and tumorigenicity, which seem to be dependent on its normal expression in several cell types. The expression of both the IGF‐I receptor and its ligands is regulated by other growth factors (especially PDGF and EGF), by oncogenes (like SV40 T antigen and c‐myb) and by tumour suppressor genes (like WT1 and RB). The picture emerging from these studies is that several transforming agents may exert their growth promoting effects through the direct or indirect activation of the IGF autocrine loop.
Liver cells can spontaneously resume proliferation in long‐term quiescent primary cultures
Authors: Lombard M.‐N. , Houssais J.‐F. , Decloitre F. , Dutrillaux B.
Published: May 2008

Abstract

We report here data on the spontaneous resumption of proliferation in long‐term primary cultures and we show that the proliferating areas are neoplastic. Normal rat hepatocytes were explanted in serum‐supplemented Ham F12 medium and maintained over 8 months without transfer. The cells remained quiescent for the first 10 weeks and they were not tumorigenic when injected into nude mice. Later, without any modification of the culture conditions or transfer, progressive changes spontaneously occurred. Foci of dividing cells were detected, some displaying y‐glutamyl‐transpep‐tidase (γ‐GT) activity and F‐actin fragmentation. These proliferating foci overcame the quiescent population. When injected into nude mice, the 15–week‐old primary cultures were highly tumorigenic, with a 3–6 week latency for tumour formation. Furthermore, a cell line was derived from a primary culture started with a liver carcinogen promoter (biliverdin‐enriched medium). This cell line proliferated rapidly and differed from a liver epithelial line, also established from our primary cultures, in its 1 karyotype (hy‐perploidy and translocation on chromosome 3), 2 requirement for arginine to proliferate, 3γ‐GT positive reaction correlated to changes in actin fibre pattern, 4 sensitivity to protease inhibitors (i.e. α2 macroglobulin, PMSF) and 5 tumorigenicity. Long‐term primary cultures and the karyotypically defined cell line are useful tools for further studies on in vitro genetic deviations.
Radiation‐induced concomitant overexpression of p53, p62c‐fos and p21N‐fas in mouse epidermis
Authors: Leszczynski D. , Servomaa K. , Lang S. , Kosmat V.‐M. , Rytömaa T.
Published: May 2008

Abstract. Early molecular events in radiation carcinogenesis in vivo are difficult to study, especially because it is usually impossible to know in advance the exact location of a radiation‐induced tumour. In the present study, we have attempted to overcome this difficulty by exposing a very small area of hairless mouse skin to high‐dose beta radiation (i.e. immobilized hot particles) on the reasonable assumption that a malignant tumour would subsequently develop and be found at the exposed site in a long‐term follow‐up. The results showed that at an exposed site, before the appearance of a visible or histologically detectable tumour, overexpression of the product of the tumour suppressor gene p53 was common (28% of the sites studied; tested with PAbl801, PAb421, PAb240 and a polyclonal antibody CM1) and that this change was regularly accompanied by overexpression of p62c‐fos and p21N‐ras. Expression of several other oncoproteins studied (p39c‐jun, p21K‐ras, p21H‐ras) was not altered at these sites. A similar pattern of changes was observed in a visible and histopathologically distinct tumour that was analyzed after its development at an exposed site. These results suggest a re'markably regular pattern of molecular changes, induced by ionizing radiation in mouse epidermis, which might be associated with carcinogenesis.
A mathematical model of glioma growth: the effect of chemotherapy on spatio‐temporal growth
Authors: Tracqui P. , Cruywagen G. C. , Woodward D. E. , Bartoo G. T. , Murray J. D. , Alvord E. C.
Published: May 2008

During the past two decades computerized tomography (CT) and magnetic resonance imaging (MRI) have permitted the detection of tumours at much earlier stages in their development than was previously possible. In spite of this earlier diagnosis the effects of earlier and more extensive treatments have been difficult to document. This failure has led to an increasing awareness of the importance of infiltration of glioma cells into surrounding grossly normal brain tissue such that recurrence still occurs. In this paper a simple mathematical model for the proliferation and infiltration of such tumours is introduced, based in part on quantitative image analysis of histological sections of a human brain glioma and especially on cross‐sectional area/volume measurements of serial CT images while the patient was undergoing chemotherapy. The model parameters were estimated using optimization techniques to give the best fit of the simulated tumour area to the CT scan data. Numerical solution of the model on a two‐dimensional domain, which took into account the geometry of the brain and its natural barriers to diffusion, was used to determine the effect of chemotherapy on the spatiotemporal growth of the tumour.
The GC factor regulates the expression of the insulin‐like growth factor‐I receptor
Authors: Morrione A. , DeAngelis T. , Baserga R.
Published: May 2008

We have transfected a plasmid expressing the transcriptional regulator GC Factor (GCF) into cell lines and have found that the GCF: 1 causes a decrease in the levels of insulin‐like growth factor I receptor (IGF‐IR) mRNA; 2 causes a decrease in the number of IGF‐IRs; and 3 represses the activity of the IGF‐IR promoter. In addition, we show that the regulation of IGF‐IR expression by GCF plays a physiological role in the control of cellular proliferation in vitro.
cAMP and PMA enhance the effects of IGF‐I in the proliferation of endometrial adenocarcinoma cell line HEC‐1‐A by acting at the G1 phase of the cell cycle
Authors: Talavera F. , Bergman C. , Pearl M. L. , Connor P. , Roberts J. A. , Menon K. M. J.
Published: May 2008

The present study was undertaken to determine whether endometrial cancer cell line HEC‐1‐A differ from nontransformed cells, in that the cAMP and protein kinase C pathways may enhance IGF‐I effects in mitogenesis by acting at the G1 phase of the cell cycle instead of G0. Immunofluorescence staining of HEC‐1‐A cells using the proliferating cell nuclear antigen (PCNA) monoclonal antibody and flow cytometric analysis determined that HEC‐1‐A cells do not enter the G0 phase of the cell cycle when incubated in a serum‐free medium. Approximately 51% of the cells were in G1, 12% were in S and 37% in G2 phase of the cell cycle prior to treatment. Forskolin and phorbol‐12‐myristate 13‐acetate (PMA) were used to stimulate cAMP production and protein kinase C activity, respectively. IGF‐I, forskolin and PMA each increased (P <0.01) [3H]‐thymidine incorporation in a dose and time dependent manner. The interaction of forskolin and PMA with IGF‐I was then determined. Cells preincubated with forskolin or PMA followed by incubation with IFG‐I incorporated significantly more (P <0.01) [3H]‐thymidine into DNA than controls or any treatment alone. It is concluded that forskolin and, to a lesser extent, PMA exert their effect at the G1 phase of the cycle to enhance IGF‐I effects in cell proliferation.
Interleukin‐3 or erythropoietin induced nuclear localization of protein kinase C β isoforms in hematopoietic target cells
Authors: Mason‐Garcia M. , Harlan R. E. , Mallia C. , Jeter J. R. , Steinberg H. B. , Fermin C. , Beckman B. S.
Published: May 2008

Protein kinase C (PKC) has been implicated in the signal transduction pathways for the biological effect of both interleukin‐3 (IL‐3) and erythropoietin (EPO) in hematopoietic target cells. The goal of this study was to identify specific classical isoforms of PKC and their localization in hematopoietic cells in response to the growth factors, IL‐3 or EPO. In addition to murine fetal liver cells as a source of normal erythroid progenitor cells, we have utilized the B6SUt.EP cell line, a non‐transformed hematopoietic cell line that requires IL‐3 for proliferation, but for which EPO can substitute as a growth factor. With polyclonal antibodies prepared against peptide sequences specific for the α, βI, βII and γ isoforms of PKC, we have identified βI and βII as the predominant nuclear isoforms in target cells that proliferate in response to IL‐3 or EPO.
Cell cycle perturbation by sodium butyrate in tumorigenic and non‐tumorigenic human urothelial cell lines assessed by flow cytometric bromodeoxyuridine/DNA analysis
Authors: Larsen J. K. , Christensen I. J. , Kieler J.
Published: May 2008

The effect of sodium butyrate on cell proliferation was studied in eight human urothelial cell lines differing in transformation grade (TGr): Hu 1752 (mortal, TGr I); HCV29 (immortal and tumorigenic, TGr II); HCV29T, T24, T24A, T24B, Hu 961A and Hu 1703He (tumorigenic, TGr III). In all cell lines, except Hu 1752, addition of 4 mm sodium butyrate at 18 h after replating resulted in a significantly decreased population of adherent cells after a further 24–48 h. This might partially be explained by detachment of cells, probably mainly S phase cells, from the substrate in the lines HCV 29, HCV29T, Hu 961A and Hu 1703He. Flow cytometric DNA analysis of the adherent cell population showed that all TGr II and III urothelial cell lines were DNA aneuploid, and that butyrate perturbed the cell cycle distribution in these cell lines, mainly by a decrease of the S phase fraction. Flow cytometric bromodeoxyuridine (BrdUrd)/DNA analysis of continuously BrdUrd labelled cultures, using a ‘washless’ BrdUrd/DNA staining technique, showed that butyrate inhibited the G0/1‐S phase transition, indicated by a delayed depletion of BrdUrd negative G0/1 cells in the cell lines HCV29, HCV29T, T24B, Hu 961A and Hu 1703He. BrdUrd/DNA analysis further showed that butyrate inhibited the G2M‐G0/1 phase transition, indicated by a pronounced accumulation of BrdUrd positive G2M cells in the cell lines HCV29T, T24B, Hu 961A and Hu 1703He. Microscopy of HCV29T and Hu 961A cells indicated that this block did not occur in mitosis. The parental cell line T 24 and the cell line T 24 A did not show an accumulation of BrdUrd negative G0/1 cells or BrdUrd positive G2M cells like that occurring in the derived cell line T 24B.
Influence of long‐term limitation of cell proliferation on the cell cycle duration
Authors: Blokhin A. V. , Khalyavkin A. V.
Published: May 2008

The hypothesis was tested that constant, long‐term inhibition of cell proliferation is the primary cause of cellular senescence. Mouse L‐929 fibroblasts were maintained in confluent cultures for periods of 6 and 12 months with cell viability maintained by regular replacement of medium and serum. The mitotic activity of the cell population under these conditions was one mitosis per 104 cells. No changes in the duration of the cell cycle were observed when, following long‐term quiescence, the cell were replated and grown at low cell density. The results do not support the hypothesis that prolonged suppression of cell proliferation induces cell senescence.
Impaired chromosome segregation in plant anaphase after moderate hypomethylation of DNA
Authors: Leyton C. , Mergudich D. , De La Torre C. , Sans J.
Published: May 2008

10−6 M and 10−5 M 5‐azacytidine, demethylated around 9% and 17% of the 5‐methylcytosine residues found in Allium cepa L. native DNA, respectively. Both treatments stimulated RNA synthesis in the cells of root meristems. On the other hand, the 10−5 M treatment gave rise to multiple chromosomal anomalies in mitosis before any fall in the mitotic index was detectable, but no chromosomal breaks were ever seen.

Serious lesions involved in chromatids and segregation in anaphase were preferentially found after hypomethylation of DNA sequences replicated in the second half of the previous S period: (i) sister telomeres remained unresolved at the cell equator while kinetochores had reached the poles, (ii) whole unsegregated chromosomes were pulled to one of the poles by obviously disfunctional kinetochores, resulting in an unbalanced distribution of chromatids, (iii) unsegregated chromosomes in other cells remained at the spindle equator as if kinetochores were nonfunctional, while cytoplasmic division took place before their migration to the poles. Frequently, a growing cytokinetic plate randomly cut the unsegregated chromosomes, giving rise to aneuploid nuclei. These anaphase failures are a firm basis to explain why the 10−5 M treatment selectively depressed the rate of cell proliferation in these cells in the long run. On the other hand, if hypomethylation occurred at the first half of the previous S period, enlarged chromosomal segments were evident in most metaphases, while chromosome laggards and bridges were recorded in anaphase at rather similar frequencies after the different 5‐azacytidine treatments. These data were consistently obtained both in the native mononucleate cells of meristems and in one subpopulation of synchronous cells labelled as binucleate by 5 mm caffeine.
Cell density downregulates DNA synthesis and proliferation during osteogenesis in vitro
Authors: Peterson W. J. , Yamaguchi D. T.
Published: May 2008

Abstract. The classical models of in vitro cell culture comprise fibroblasts and epithelial cells. Osteogenic cells represent another interesting cell model; however, it is not known whether during osteogenesis cell density regulates cell growth as seen in cultures of fibroblasts and epithelial cells. We selected MC3T3‐E1 cells for study because they are an osteogenic cell line that, when subcultured, grow to confluence and form multilayers of cells in conventional cultures by continued proliferation, as do fibroblasts. Once maximum cell density is obtained, proliferation is down regulated resulting in a mixed population of quiescent and dividing cells. We used this model to determine whether downregulation of proliferation as expressed by cell number and DNA synthesis is cell density‐dependent. MC3T3‐E1 cells were cultured over a period of 34 days to determine their kinetics, viability, ability to synthesize DNA, distribution within phases of the cell cycle and cell number‐response relationships. Our results show that (1) viability ranged between 92% and 96% and the cell number 2.5 x 105 per cm2 once cultures reached steady state, (2) most cells entered the G0/G1 phase of the cell cycle on day 7, (3) there was no correlation between the proportion of cells in S phase and downregulation of DNA synthesis, (4) a direct relationship exists between cell density and downregulation of DNA synthesis on day 8, (5) the minimum time for cells to be cultured before downregulation of DNA synthesis begins is independent of cell number, and (6) downregulation of DNA synthesis is reversible. These results suggest that density‐dependent downregulation of DNA synthesis may be a mechanism of growth control for osteogenic cells in vitro that operates more like density‐dependent growth control in cultures of fibroblasts rather than epithelial cells.
Characterization of label‐retaining cells in the epidermis of a human skin equivalent
Authors: Graeter L. J. , Hull B. E.
Published: May 2008

Abstract. The human skin equivalent (HSE) is an in vitro reconstructed model that resembles skin morphologically and biochemically. The HSE is formed by overlaying a fibroblast‐populated collagen matrix with a suspension of epidermal cells. Basal keratinocytes attach to the dermal equivalent via a newly formed basement membrane and multiply to form a stratified, differentiated epidermis. The aim of the studies described here was to characterize the basal cells of the HSE in terms of their cell cycling potential. The experiments utilized long‐term labelling of the cells with tritiated thymidine ([3H]dT), followed by irradiation with ultraviolet light. [3H]dT incorporation was analysed via routine autoradiography. Irradiation with 100 J/m2 UV light increased the number of labelled basal cells by 58% over the control, the maximal stimulation observed. Decreased numbers of labelled basal cells were observed at doses of UV light greater than 100 J/m2. The maximal number of labelled basal cells was observed on day 14 and decreased over time; the number of labelled suprabasal cells increased concomitantly. Label‐retaining cells (12%) persisted in the stratum basale of control HSEs after 32 days in culture. Labelled cells were observed in the apical layers of the stratum granulosum of control HSEs after 22 days in culture. These data suggest that the stratum basale of the HSE contains a population of slow‐cycling cells whose characteristics resemble a subpopulation of slowly cycling cells found in normal human skin.
c‐erbB‐2/neu protein expression, DNA ploidy and S phase in breast cancer
Authors: Dittadi R. , Calderazzo F. , Cabrelle A. , Fresco S. Di , Gion M. , Chieco‐Bianchi L.
Published: May 2008

Abstract. DNA content and c‐erbB2/neu protein (p185) expression were evaluated by flow‐cytometry and ELISA, respectively, in 166 specimens of primary breast cancer. A non‐diploid DNA content was found in 88 tumours (53%), with the DNA index ranging from 0.7‐2.7. S phase fraction (SPF) evaluation, performed in 130 cases, showed significantly higher values in aneuploid than in diploid tumours (median values, 17.3% and 5.8%, respectively). Thirty‐six tumours (21.6%) showed p185 overexpression, while 45 (27.1%) and 85 (51.3%) showed intermediate and low expression, respectively. A good correlation (P=0.0023) was found between DNA content and p185 positivity. Tumours with high p185 values were mainly aneuploid, while tumours with intermediate or low expression had variable degrees of DNA content. Furthermore, p185 concentration was significantly higher in aneuploid than in diploid tumours (P=0.009). The highest rate of p185 (+) cases and the highest p185 concentrations occurred in triploid (1.3<D.I≦1.7), compared to the other tumours. SPF values and p185 positivity rates were not significantly correlated in diploid and in aneuploid tumours.
Region‐specific DNA synthesis in brains of F344 rats following a six‐day bromodeoxyuridine infusion
Authors: Bolon B. , Dunn C. , Goldsworthy T. L.
Published: May 2008

Abstract. Prolonged exposure to certain alkylating chemicals induces glial and meningeal tumours in rats, probably resulting from DNA damage to dividing neural cells. The present work evaluated DNA synthesis in the brains of untreated, young adult male F344 rats in order to define a BrdUrd infusion protocol to more adequately assess proliferation in slowly dividing neural cell populations. BrdUrd (2.5 to 160 mg/ml) was administered for 6 days via subcutaneous osmotic pumps. Clinical toxicity was not observed at any dose. The labelling index (LI; % of cells per brain area that incorporated BrdUrd) and unit length labelling index (ULLI; % of cells per meningeal length that incorporated BrdUrd) were calculated for selected regions by counting labelled neural cells in defined areas of the right hemisphere in coronal brain sections. Intensely stained cells were numerous in the cerebral subependymal layer (LI=35.8%); scattered in cerebral white matter tracts (e.g. corpus callosum and internal capsule; LI=6.2%) as well as cerebral (ULLI=4.2%) and cerebellar (ULLI=3.6%) meninges; and rare in the hippocampus (LI>0.1%). Mildy stained cells were dispersed in the pons (LI=2.1%), deep cerebral (LI=1.8%) and cerebellar (LI=1.0%) grey matter, and thalamus (LI=0.3%). Phenotypically, BrdUrd‐positive cells in neuropil were glial cell precursors and their progeny, while those associated with meninges were usually located in the superficial subarachnoid space and appeared to be fibrocytes. Using BrdUrd infusion, LI for glial precursors at these sites ranged from two‐ to 10‐fold higher than those reported previously after a brief parenteral pulse dose. These data indicate that continuous BrdUrd infusion for 6 days by subcutaneous osmotic pump is an efficient means of labelling neural cells throughout the brain.
Differential effects of 2′‐deoxyguanosine on peripheral blood mononuclear cell proliferation in healthy donors and Hashimoto's thyroiditis patients
Authors: Covas M. I. , Esquerda A. , Arner M. , Sanz F. , Mahy N.
Published: May 2008

Abstract. The aim of this study was to determine possible differences in peripheral blood mononuclear cells (PBMC) proliferation of healthy donors and Hashimoto's thyroiditis patients and whether a statistical approach to cell proliferation analysis might be used to discern the differences. The effect of a wide range of 2′‐deoxyguanosine (dGuo) concentrations (0–1250 μM) on the mitogen‐induced proliferation of PBMC was studied in healthy donors and Hasimoto's thyroiditis patients. Activity levels of purine nucleoside phosphorylase (PNP) and adenosine deaminase (ADA) in PBMC were also measured. For the first time in a study of these models of dGuo toxicity in vitro, the analysis of polynomial trends of orders from 1 to 7 was applied to evaluate cell proliferation. A dose‐dependent inhibition of mitogen‐induced PBMC proliferation was observed in both groups. Data for linear trend established that PBMC from Hashimoto's thyroiditis patients were more sensitive to dGuo toxicity than PBMC from healthy donors. A positive quadratic trend at low dGuo doses was found in the cell proliferation of Hashimoto's thyroiditis patients. A decrease in PNP activity (P < 0.025) and an increase in ADA activity (P < 0.005) was observed in PBMC of Hashimoto's thyroiditis group. The differences in PBMC proliferation subjected to dGuo toxicity between the two groups could be related with the distinct pattern of purine salvage enzymes observed.
The effects of irradiation at different times of the day on rat intestinal goblet cells
Authors: Becciolini A. , Balzi M. , Fabbrica D. , Potten C. S.
Published: May 2008

Quantitative changes in jejunal goblet cells were studied in control and whole body irradiated rats using PAS‐Alcian blue staining of crypt sections. A circadian dependence was observed when control animals were killed at different times during the light/dark cycle. Irradiation with 3 Gy produced a 2–3‐fold increase within 36 h in goblet cells relative to controls, followed by a reduction to very low levels. There was a return to pre‐treatment levels later than was observed for the columnar cells. The present results on the pattern of response of goblet cells and those of brush border enzyme activity are consistent with the hypothesis that ionizing radiation can influence differentiation. In fact during the first hours after irradiation an early induction of differentiation is evident while during the early repopulation phase columnar cells prevailed relative to the goblet cells. Only at later times were normal differentiation patterns seen. Groups of animals exposed to the same dose of radiation at different times of the day showed similar general patterns of behaviour even if the group irradiated at midnight showed a more marked and longer lasting injury.
Inhibition of Tobacco Mosaic Virus Reproduction in Isolated Tobacco Protoplasts by Means of Pancreatic Ribonuclease
Authors: Zhuravlev Yu. N. , Pisetskaya N. F. , Ledneva V. A.
Published: May 2008

Treatment of protoplasts with RNase solutions (1 — 100 μg/ml) sharply reduced their ability to support TMV reproduction. Treatment of protoplasts with RNase both prior to and after inoculation with the virus, and also treatments of varying duration, had approximately the same effect on inhibiting TMV reproduction; however, the effect became sharply intensified with higher RNase concentrations. The authors assume that the RNase inhibitive effect in protoplasts is ensured by the specific structure of this relatively small positively‐charged macromolecule, not by its enzymic properties. The RNase molecules apparently have a damaging effect on the plasmalemma and possibly on other cell membranes with which the initial stages of TMV reproduction are associated. The development of the RNase damaging effect can be prevented by bivalent metal ions in the incubation medium, especially by calcium and manganese. The protective effect of cations is presumably caused not only by the charge, but by their steric configuration since K+ and Mg++ are ineffective in protecting the protoplasts.
Incidence and Control of Necrotic Leaf Mosaic Caused by Arabis Mosaic Virus in Lilium tigrinum splendens in the Netherlands
Authors: Asjes C. J. , Segers Louise Ch.
Published: May 2008

Necrotic mosaic on leaves and ring spots on bulb scales of Lilium tigrinum splendens, can be caused by arabis mosaic virus (AMV). Primarily infected bulbs can show spongy roots and large necrotic areas on creamy coloured bulb scales. Consecutive series of plants replanted for monthly periods in infested soil were mostly infected by AMV at a high rate (70%) throughout the year. Very low or undetectable numbers of Xiphinema diversicandatum, nematodes in soil dilution experiments infected lilies very efficiently (50–70 %). In general, soil disinfestation with dichloropropene, dazomet, methylbromide, and other disinfectants were variably fairly effective, particularly when yellow crocus among which couch was abundant, was previously grown for two years. The influx of AMV infected material into the soil was assumed to increase the number of AMV‐carrying nematodes, and may be one cause of the failure of soil disinfestation. A survey of AMV infested soil in lily‐growing regions in The Netherlands indicated its occurrence in a few fields only. Complex control measures applicable under growers' conditions, in addition to the variably effective soil disinfestation, are indicated.