Exponential Cell Growth Research Articles

Sparc localizes to the blebs of hobit cells and human primary osteoblasts

Secreted protein acidic and rich in cystein (SPARC) is a secreted glycoprotein involved in several biological processes such as tissue remodeling, embryonic development, cell/extracellular matrix interactions, and cell migration. In particular, SPARC affects bone remodeling through the regulation of both differentiation/survival of osteoblasts and bone extracellular matrix synthesis/turnover. Here, we investigated SPARC subcellular localization in the human osteoblastic HOBIT cell line by immunocytochemistry and western blot analysis. We show that, under normal exponential cell growth conditions, SPARC localized both to cell nucleus and to cytoplasm, with no co-localization on actin stress fibers. However, in colchicine-treated HOBIT cells and human primary osteoblasts undergoing blebs formation, SPARC showed a different cellular distribution, with an additional marked compartmentalization inside the blebs, where it co-localized with globular actin and actin-binding proteins such as alpha-actinin, cortactin, and vinculin. Moreover, we demonstrate by an in vitro assay that the addition of SPARC to actin and alpha-actinin inhibited the formation of cross-linked actin filaments and disrupted newly formed filaments, most likely due to a direct interaction between SPARC and alpha-actinin, as indicated by immunoprecipitation assay. The specific silencing of SPARC RNA expression markedly decreased the ability of colchicine-treated HOBIT cells to undergo blebbing, suggesting a direct role for SPARC in cell morphology dynamics during cytoskeletal reorganization.

Metabolic flux model for an anchorage‐dependent MDCK cell line: Characteristic growth phases and minimum substrate consumption flux distribution

Up to now cell-culture based vaccine production processes only reach low productivities. The reasons are: (i) slow cell growth and (ii) low cell concentrations. To address these shortcomings, a quantitative analysis of the process conditions, especially the cell growth and the metabolic capabilities of the host cell line is required. For this purpose a MDCK cell based influenza vaccine production process was investigated. With a segregated growth model four distinct cell growth phases are distinguished in the batch process. In the first phase the cells attach to the surface of the microcarriers and show low metabolic activity. The second phase is characterized by exponential cell growth. In the third phase, preceded by a change in oxygen consumption, contact inhibition leads to a decrease in cell growth. Finally, the last phase before infection shows no further increase in cell numbers. To gain insight into the metabolic activity during these phases, a detailed metabolic model of MDCK cell was developed based on genome information and experimental analysis. The MDCK model was also used to calculate a theoretical flux distribution representing an optimized cell that only consumes a minimum of carbon sources. Comparing this minimum substrate consumption flux distribution to the fluxes estimated from experiments unveiled high overflow metabolism under the applied process conditions.

Organic carbon, and not copper, controls denitrification in oxygen minimum zones of the ocean

Incubation experiments under trace metal clean conditions and ambient oxygen concentrations were used to investigate the response of microbial assemblages in oxygen minimum zones (OMZs) to additions of organic carbon and copper, two factors that might be expected to limit denitrification in the ocean. In the OMZs of the Eastern Tropical North and South Pacific, denitrification appeared to be limited by organic carbon; exponential cell growth and rapid nitrate and nitrite depletion occurred upon the addition of small amounts of carbon, but copper had no effect. In the OMZ of the Arabian Sea, neither carbon nor copper appeared to be limiting. We hypothesize that denitrification is variable in time and space in the OMZs in ways that may be predictable based on links to the episodic supply of organic substrates from overlying productive surface waters.

The risk to develop a recurrence of a gastric cancer—is it independent of time?

Recurrence is the main reason for early death of cancer patients. Therefore, survival curves are regarded as crucial tools for clinicians to evaluate therapies and to estimate the patients' prognosis. Current models for the development of recurrence are based on the assumption of a residual cancer cell burden after therapy. Accordingly, in assumption of an exponential cell growth of the cancer cells an S-shaped decline of the survival curve is expected with earlier onset in case of advanced cancer and a later manifestation in case of little residual tumour. However, many survival curves do not reflect any S-shaped configuration and thus may question the current concept. To test, whether the incidence for developing a recurrence may be considered as remaining constant over time, we analysed survival data of 446 patients with gastric cancer, operated from 1975-2001 in the Surgical Department of the RWTH Aachen. All survival curves, even after sub-grouping according to UICC stage, show a monotonous decline without any apparent S-shape. The impact of TNM and UICC stage to predict the survival in patients is estimated by Cox regression, for estimation the risk for death a logistic regression is performed. Whereas the presence of metastasis lowers the prognosis significantly with a hazard ratio of 1.57 and an odds ratio of 7.56, respectively, a significant relevance for the UICC stage, the tumour size or the lymph node status cannot be proven. Furthermore, the two assumptions: (1) that 20% of patients who are still alive after 5 years have been cured, and (2) that the remainder develop a recurrence in constantly 7.3% per month, are able to configure the survival curve almost precisely (correlation coefficient between calculated and observed survival rate r > 0.99). The absence of any S-shaped survival curve configuration is not in accordance with the focus on residual tumour clones with its exponential growth as the decisive process for recurrence development. In contrast, the monotonous decline of surviving patients is best reflected by a constant incidence of recurrence. The (time) constancy of the recurrence incidence encourages the view of recurrent cancer as a chronic problem of a carcinogenic environment. Furthermore, it supports new anticancer therapies which rather targets cell regulation and immunology instead of acting cytotoxic.

Glutathione Is Recruited into the Nucleus in Early Phases of Cell Proliferation

We have studied the possible correlation between nuclear glutathione distribution and the progression of the cell cycle. The former was studied by confocal microscopy using 5-chloromethyl fluorescein diacetate and the latter by flow cytometry and protein expression of Id2 and p107. In proliferating cells, when 41% of them were in the S+G(2)/M phase of the cell cycle GSH was located mainly in the nucleus. When cells reached confluence (G(0)/G(1)) GSH was localized in the cytoplasm with a perinuclear distribution. The nucleus/cytoplasm fluorescence ratio for GSH reached a maximal mean value of 4.2 +/- 0.8 at 6 h after cell plating. A ratio higher than 2 was maintained during exponential cell growth. In the G(0)/G(1) phase of the cell cycle, the nucleus/cytoplasm GSH ratio decreased to values close to 1. We report here that cells concentrate GSH in the nucleus in the early phases of cell growth, when most of the cells are in an active division phase, and that GSH redistributes uniformly between the nucleus and the cytoplasm when cells reach confluence.

Functional analysis of the gene expression profiles of colorectal cancer cell lines in relation to oxaliplatin and cisplatin cytotoxicity

The objective was to relate the gene expression profiles of colorectal cancer cells in culture to the in vitro cytotoxicity of cisplatin and oxaliplatin. We studied the gene expression profiles of six human colorectal cancer cell lines, using the Atlas Plastic Human 8K Microarray from Clontech, and related it to the in vitro cytotoxicities of oxaliplatin and cisplatin obtained by inhibition of exponential growth of cells. We calculated the Pearson's coefficients of correlation (r) between gene expression and drug IC50. A functional analysis was performed using the Gene Ontology Consortium database. Results were validated on a series of representative genes by real-time quantitative PCR. Validation of the significance of the coefficients of correlation was also performed using a leave-one-out analysis. We identified 394 genes whose expression was significantly correlated (P<0.05) to oxaliplatin cytotoxicity and 40 with cisplatin cytotoxicity. Three major functions were preferentially involved in oxaliplatin activity: protein synthesis, cell energetics and response to oxidative stress. No significant correlation was observed between oxaliplatin or cisplatin cytotoxicity and the expression of genes involved in DNA repair, cell proliferation or cell adhesion. A strongly significant correlation was found between the microarray and the rt-PCR approaches (r=0.968, P<10(-6)). The leave-one-out analysis showed that the same functions still appeared significantly involved in the activity of both drugs. Based on the functional analysis, we hypothesized that oxaliplatin would specifically form protein adducts during synthesis, thus exposing their thiol groups, which are known to be especially vulnerable to reactive oxygen species.

Fed-batch culture optimization of a growth-associated hybridoma cell line in chemically defined protein-free media

An investigation was made to study the processes of fed-batch cultures of a hybridoma cell line in chemically defined protein-free media. First of all, a strong growth-associated pattern was correlated between the production of MAb and growth of cells through the kinetic studies of batch cultures, suggesting the potential effectiveness of extending the duration of exponential growth in the improvement of MAb titers. Second, compositions of amino acids in the feeding solution were balanced stepwisely according to their stoichiometrical correlations with glucose uptake in batch and fed-batch cultures. Moreover, a limiting factor screening revealed the constitutive nature of Ca(2+) and Mg(2+) for cell growth, and the importance of their feeding in fed-batch cultures. Finally, a fed-batch process was executed with a glucose uptake coupled feeding of balanced amino acids together with groups of nutrients and a feeding of CaCl(2) and MgCl(2) concentrate. The duration of exponential cell growth was extended from 70 h in batch culture and 98 h in fed-batch culture without Ca(2+)/Mg(2+) feeding to 117 h with Ca(2+)/Mg(2+) feeding. As a result of the prolonged exponential cell growth, the viable and total cell densities reached 7.04 x 10(6) and 9.12 x 10(6) cells ml(-1), respectively. The maximal MAb concentration achieved was increased to approximately eight times of that in serum supplemented batch culture.

Modelling Cell Growth and its Modulation of the G1/S Transition

We present a model for the regulation of the G(1)/S transition by cell growth in budding yeast. The model includes a description of cell size, the extracellular nutrient concentration and a simplified model of the G(1)/S transition as originally reported by Chen et al. [Mol. Biol. Cell 11:369-391, 2000]. By considering cell growth proportional to cell size we show that the cell grows exponentially. In the case where cell growth is considered proportional to the concentration of a sizer protein within the cell, our model exhibits both exponential and linear cell growth for varying parameter values. The effects of varying nutrient concentration and initial cell size are considered in the context of whether progression through the cell-size checkpoint occurs. We consider our results in relation to recent experimental evidence and discuss possible experiments for testing our theoretical predictions.

Contribution of alginate and levan production to biofilm formation by Pseudomonas syringae

Exopolysaccharides (EPSs) play important roles in the attachment of bacterial cells to a surface and/or in building and maintaining the three-dimensional, complex structure of bacterial biofilms. To elucidate the spatial distribution and function of the EPSs levan and alginate during biofilm formation, biofilms of Pseudomonas syringae strains with different EPS patterns were compared. The mucoid strain PG4180.muc, which produces levan and alginate, and its levan- and/or alginate-deficient derivatives all formed biofilms in the wells of microtitre plates and in flow chambers. Confocal laser scanning microscopy with fluorescently labelled lectins was applied to investigate the spatial distribution of levan and an additional as yet unknown EPS in flow-chamber biofilms. Concanavalin A (ConA) bound specifically to levan and accumulated in cell-depleted voids in the centres of microcolonies and in blebs. No binding of ConA was observed in biofilms of the levan-deficient mutants or in wild-type biofilms grown in the absence of sucrose as confirmed by an enzyme-linked lectin-sorbent assay using peroxidase-linked ConA. Time-course studies revealed that expression of the levan-forming enzyme, levansucrase, occurred mainly during early exponential growth of both planktonic and sessile cells. Thus, accumulation of levan in biofilm voids hints to a function as a nutrient storage source for later stages of biofilm development. The presence of a third EPS besides levan and alginate was indicated by binding of the lectin from Naja mossambica to a fibrous structure in biofilms of all P. syringae derivatives. Production of the as yet uncharacterized additional EPS might be more important for biofilm formation than the syntheses of levan and alginate.

Effects of medium composition on production of 5-aminolevulinic acid by recombinant Escherichia coli

The recombinant Escherichia coli BL21(DE3) harboring hemA from Agrobacterium radiobacter, which was engineered in our previous work, was used for the extracellular production of 5-aminolevulinic acid (ALA). The effects of various physiological factors, such as the concentrations of precursors (glycine, succinic acid and glucose) and the inhibitor 5-aminolevulinate dehydratase (levulinic acid), on the ALA accumulation in the fermentation broth were investigated in both shake flasks and a jar fermentor. Among these precursors, glycine exhibited the strongest ability to inhibit cell growth, while glucose mainly inhibited ALA formation. The optimum initial concentrations of glycine, succinic acid and glucose were found to be 2.0, 10.0 and 2.0 g/l, respectively. Levulinic acid (LA; 30 mM) was fed to the fermentation broth at the end of the exponential cell growth phase (about 8 h), and the intracellular activity of ALA dehydratase was efficaciously suppressed. Repeating the optimum composition of the medium in a stirred tank fermenter resulted in 1.49 g/l ALA. Furthermore, the fed batch of the precursors and inhibitor further increased ALA production up to 3.01 g/l.

Self‐assembled copper‐capillary alginate gel scaffolds with oligochitosan support embryonic stem cell growth

Biomaterial scaffolds are fundamental components of strategies aimed at engineering a wide range of tissues. Scaffolds possessing uniform, oriented microtubular architectures could be ideal for multiple tissues, but are challenging to produce. Therefore, we developed hydrogel scaffolds possessing regular, tubular microstructures from self-assembled copper-capillary alginate gel (CCAG). To abrogate the rapid dissolution of CCAG in cell culture media, we treated it with oligochitosan and created a stable oligochitosan-CCAG (OCCAG) polyelectrolyte complex. Fourier transform infrared spectroscopy confirmed polyelectrolyte complexation between alginate and oligochitosan. OCCAG retained capillary morphology, shrank anisotropically in bulk, lost Cu(2+) ions, and maintained (71.9 +/- 5.65)% of its mass in cell culture media. Next, we seeded mouse embryonic stem (ES) cells within OCCAG scaffolds, and examined cell morphology and quantified cell growth and viability over four days. ES cells were guided to form cylindrical structures of staggered cells within scaffold capillaries. Analysis of the total cells recovered from the scaffolds revealed exponential cell growth (normalized to day 0) that was statistically similar to gelatinized-plate controls. OCCAG-cultured ES cell viability was also not significantly different from controls at day 4. CCAG-derived scaffolds can therefore serve as a unique platform for stem cell-based tissue engineering.

Cancer and Evolution

Cancer mostly is a disease of old age. Evolutionary pressures have pushed the somatic "error rate", especially the mutation rate, down to a level where for most organisms cancer is no longer of any selective significance. This appears to be a by-product of the selection that gives rise to senescence, following the arguments of Medawar, Holliday, and Kirkwood. The development of a cancer is discussed from an evolutionary viewpoint, emphasising the role of selection versus mutation and the fact that each cancer is an independent evolutionary process. The nature of the selective advantages associated with the somatic genetic changes during a cancer's evolution can sometimes be inferred by reference to the known types of mutations found in cancers. Examples are given using colorectal cancer as a model. The major selective forces involve the balance between selection for increased growth rate and against apoptosis. There are strong arguments against the much discussed role of genomic instability as a requirement for cancer. Current evidence suggests that instability is a byproduct of selection against apoptosis. There is an important contrast between germ line and somatic changes, the former being the basis for inherited susceptibilities to cancer, while the latter are the fundamental changes that turn a normal cell into a cancer cell. Tissue stem cells, as in the colonic crypt, provide the source, through division and differentiation, of the differentiated cells in a crypt. Mathematical models can provide an explanation for how the balance in a crypt between stem cells, intermediate proliferating cells, and non-proliferating differentiated cells is maintained. Perturbations of the renewal parameters in the model can explain the evolution of benign tumors, namely polyps or adenomas, and the eventually exponential growth of cells resulting in a fully developed carcinoma. It seems probable that the origin of most carcinomas is in the intermediate proliferating cells, and that these are therefore the likely source of cancer stem cells.

Distinguishing between linear and exponential cell growth during the division cycle: Single-cell studies, cell-culture studies, and the object of cell-cycle research

BackgroundTwo approaches to understanding growth during the cell cycle are single-cell studies, where growth during the cell cycle of a single cell is measured, and cell-culture studies, where growth during the cell cycle of a large number of cells as an aggregate is analyzed. Mitchison has proposed that single-cell studies, because they show variations in cell growth patterns, are more suitable for understanding cell growth during the cell cycle, and should be preferred over culture studies. Specifically, Mitchison argues that one can glean the cellular growth pattern by microscopically observing single cells during the division cycle. In contrast to Mitchison's viewpoint, it is argued here that the biological laws underlying cell growth are not to be found in single-cell studies. The cellular growth law can and should be understood by studying cells as an aggregate.ResultsThe purpose or objective of cell cycle analysis is presented and discussed. These ideas are applied to the controversy between proponents of linear growth as a possible growth pattern during the cell cycle and the proponents of exponential growth during the cell cycle. Differential (pulse) and integral (single cell) experiments are compared with regard to cell cycle analysis and it is concluded that pulse-labeling approaches are preferred over microscopic examination of cell growth for distinguishing between linear and exponential growth patterns. Even more to the point, aggregate experiments are to be preferred to single-cell studies.ConclusionThe logical consistency of exponential growth – integrating and accounting for biochemistry, cell biology, and rigorous experimental analysis – leads to the conclusion that proposals of linear growth are the result of experimental perturbations and measurement limitations. It is proposed that the universal pattern of cell growth during the cell cycle is exponential.

MicroRNAs 221 and 222 inhibit normal erythropoiesis and erythroleukemic cell growth via kit receptor down-modulation

MicroRNAs (miRs) are small noncoding RNAs that regulate gene expression primarily through translational repression. In erythropoietic (E) culture of cord blood CD34+ progenitor cells, the level of miR 221 and 222 is gradually and sharply down-modulated. Hypothetically, this decline could promote erythropoiesis by unblocking expression of key functional proteins. Indeed, (i) bioinformatic analysis suggested that miR 221 and 222 target the 3' UTR of kit mRNA; (ii) the luciferase assay confirmed that both miRs directly interact with the kit mRNA target site; and (iii) in E culture undergoing exponential cell growth, miR down-modulation is inversely related to increasing kit protein expression, whereas the kit mRNA level is relatively stable. Functional studies show that treatment of CD34+ progenitors with miR 221 and 222, via oligonucleotide transfection or lentiviral vector infection, causes impaired proliferation and accelerated differentiation of E cells, coupled with down-modulation of kit protein: this phenomenon, observed in E culture releasing endogenous kit ligand, is magnified in E culture supplemented with kit ligand. Furthermore, transplantation experiments in NOD-SCID mice reveal that miR 221 and 222 treatment of CD34+ cells impairs their engraftment capacity and stem cell activity. Finally, miR 221 and 222 gene transfer impairs proliferation of the kit+ TF-1 erythroleukemic cell line. Altogether, our studies indicate that the decline of miR 221 and 222 during exponential E growth unblocks kit protein production at mRNA level, thus leading to expansion of early erythroblasts. Furthermore, the results on kit+ erythroleukemic cells suggest a potential role of these miRs in cancer therapy.

Establishment and Characterization of a Novel Human Pancreatic Cancer Cell Line (SUIT-4) Metastasizing to Lymph Nodes and Lungs in Nude Mice

A new tumor cell line (SUIT-4) derived from ascites of a patient with carcinoma of the pancreas has been established in tissue culture and in nude mice, and maintained for over 7 years. In tissue culture, the cells grew as a confluent monolayer with piling up of cells in some areas. The population doubling time during the exponential phase of the cell growth was 43.9 h in vitro. Chromosome count ranged from 63 to 68 with a modal number of 67. Subcutaneous injection of cultured cells into the flanks of nude mice resulted in tumor formation with a doubling time of 88.8 h. Histopathologically, xenografts in nude mice were moderately differentiated tubular adenocarcinoma, and the tumor cells showed spontaneous metastasis to the regional lymph nodes in 6 of 21 nude mice and to the lung in 4 of 21. Transmission electron microphotographs confirmed the ductal cell origin of the carcinoma and revealed that the cells had abundant mitochondria and lysosomes. SUIT-4 cells released carcinoembryonic antigen (3.08 × 10² ng/1 × 10⁶ cells/24 h) and carbohydrate antigen 19-9 (4.75 × 10⁴ U/1 × 10⁶ cells/24 h) during exponential cell growth in vitro. Reverse transcriptase-polymerase chain reaction studies revealed that SUIT-4 cells expressed matrix metalloproteinases 1, 3, 7, 10 and 14.

The Reduction of P-Glycoprotein Expression by Small Interfering RNAs Is Improved in Exponentially Growing Cells

Small interfering RNAs (siRNAs) are powerful tools in specifically silencing gene expression. Nevertheless, their efficiency can be limited when targeting proteins with an unusually long half-life, such as P-glycoprotein (P-gp), which is involved in the multidrug resistance phenomenon. P-gp is characterized by a long half-life, which may vary depending on the cell line and, for some of them, on serum deprivation or high cell density. In the present paper, involvement of an exponential cell growth phase in the improvement of siRNA efficiency has been suggested. The doxorubicin-selected human line MCF7-R was shown to be a more adapted model than NIH-MDR-G185 cells stably transfected with human mdr1. Nonspecific effects occurring at moderate (100 nM) siRNA concentration have been shown. Two efficient siRNAs led to a very satisfactory P-gp extinction (only 20% P-gp expression remaining) with siRNA concentration as low as 20 nM.

Glutathione Regulates Telomerase Activity in 3T3 Fibroblasts

Changes in telomerase activity have been associated either with cancer, when activity is increased, or with cell cycle arrest when it is decreased. We report that glutathione, a physiological antioxidant present at high intracellular concentrations, regulates telomerase activity in cells in culture. Telomerase activity increases in 3T3 fibroblasts before exponential cell growth. The peak of telomerase activity takes place 24 h after plating and coincides with the maximum levels of glutathione in the cells. When cells are treated with buthionine sulfoximine, which decreases glutathione levels in cells, telomerase activity decreases by 60%, and cell growth is delayed. Glutathione depletion inhibits expression of E2F4 and Id2, which regulate the cell cycle. When glutathione levels are restored after incubation with glutathione monoethylester, telomerase activity and the cell cycle-related proteins return to control values. To discover the effect of glutathione redox status on the telomerase multicomplex structure, we incubated protein extracts from fibroblasts with different glutathione redox buffers. Telomerase activity is maximal under reduced conditions i.e. when the reduced/oxidized glutathione ratio is high. Consequently glutathione concentration parallels telomerase activity. These results underscore the main role of glutathione in the control of telomerase activity and of the cell cycle.

Influence of culture conditions on the production of extra-cellular 5-aminolevulinic acid (ALA) by recombinant E. coli

Extra-cellular production of 5-aminolevulinic acid (ALA) by recombinant E. coli harboring a fusion gene hemA:egfp was investigated in shake flasks and a bioreactor with chemically defined media by varying physiological factors such as the concentrations of precursors (glycine and succinic acid), and levulinic acid (LA). Optimum concentrations of glycine and succinic acid were found to be 15 and 30 mM, respectively. When 30 mM LA was added to the culture medium at the end of the exponential cell growth phase, intra-cellular ALA dehydratase activity was suppressed. Some imbalances might be caused by the addition of titrants to the cultivation. Therefore, the recombinant E. coli used in this study were grown with no control over the culture pH in the beginning phase. The recombinant E.coli produced significant ALA in the initial pH ranges of 6.1 and 6.5, i.e. without the addition of titrants. IPTG induction for the expression of the fusion gene did not enhance the production of extra-cellular ALA. Repeated addition of glycine, succinic acid, and LA increased the production of extra-cellular ALA and the inhibition of intra-cellular ALA activity, with up to 1.3 g/l ALA having been produced in the cultivation.

B-Myb acts as a repressor of human COL1A1 collagen gene expression by interacting with Sp1 and CBF factors in scleroderma fibroblasts

We investigated the role of B-Myb, a cell-cycle-regulated transcription factor, in the expression of the alpha1 (I) pro-collagen gene (COL1A1) in scleroderma fibroblasts. Scleroderma or SSc (systemic sclerosis) is a fibrotic disease characterized by excessive production of extracellular matrix components, especially type I collagen. Northern-blot analysis showed an inverse relationship between COL1A1 mRNA expression and that of B-Myb during exponential cell growth and during quiescence in human SSc fibroblasts. Overexpression of B-Myb in SSc fibroblasts was correlated with decreased COL1A1 mRNA expression. Transient transfections localized the down-regulatory effect of B-Myb to a region containing the proximal 174 bp of the COL1A1 promoter that does not contain B-Myb consensus binding sites. Gel-shift analysis, using nuclear extracts from normal and SSc fibroblasts transfected with B-Myb, showed no differences in DNA-protein complex formation when compared with the nuclear extracts from mock-transfected cells. However, we found that B-Myb decreases Sp1 (specificity protein 1) and CBF (CCAAT-binding factor) binding for their specific sites localized in the 174 bp COL1A1 proximal promoter. These results were also confirmed using B-Myb-immunodepleted nuclear extracts. Furthermore, immunoprecipitation assays using SSc nuclear extracts demonstrated a physical interaction of B-Myb with Sp1 and CBF transcription factors, and also an interaction between Sp1 and CBF. In addition, by employing full-length or deleted B-Myb cDNA construct, we found that B-Myb down-regulates the COL1A1 proximal promoter through its C-terminal domain. Thus these results suggest that B-Myb may be an important factor in the pathway(s) regulating collagen production in SSc fibroblasts.

Dynamics of subgenomic hepatitis C virus replicon RNA levels in Huh-7 cells after exposure to nucleoside antimetabolites.

Treatment with antimetabolites results in chemically induced low nucleoside triphosphate pools and cell cycle arrest in exponentially growing cells. Since steady-state levels of hepatitis C virus (HCV) replicon RNA were shown to be dependent on exponential growth of Huh-7 cells, the effects of antimetabolites for several nucleoside biosynthesis pathways on cell growth and HCV RNA levels were investigated. A specific anti-HCV replicon effect was defined as (i). minimal interference with the exponential cell growth, (ii). minimal reduction in cellular host RNA levels, and (iii). reduction of the HCV RNA copy number per cell compared to that of the untreated control. While most antimetabolites caused a cytostatic effect on cell growth, only inhibitors of the de novo pyrimidine ribonucleoside biosynthesis mimicked observations seen in confluent replicon cells, i.e., cytostasis combined with a sharp decrease in replicon copy number per cell. These results suggest that high levels of CTP and UTP are critical parameters for maintaining the steady-state level replication of HCV replicon in Huh-7 cells.