Cellular ras activity and phospholipid metabolism

Cellular ras activity has been neutralized in 3T3 cells by microinjection of a specific anti-ras monoclonal antibody. The injected antibody efficiently inhibited proliferation in cells treated with a phorbol ester and a calcium ionophore, or with prostaglandin F2 alpha. These treatments were designed to imitate the action of phospholipase C or of phospholipase A2. In addition, the highly efficient mitogenic potential of phosphatidic acid was inhibited by the injected antibody even more efficiently than was serum-induced proliferation. The close reliance of phospholipid-induced mitogenesis upon ras activity suggests that ras proteins are unlikely to function to control the action of a phospholipase.     

Role of the isoprenoid pathway in ras transforming activity, cytoskeleton organization, cell proliferation and apoptosis

The isoprenoid pathway, by its end-products lipids, has an important role in the control of ras transforming activity, cytoskeleton organization, cell proliferation and apoptosis. In particular, prenylation, a post-translational addition of farnesol and geranylgeraniol, is an important event for the biological activity of many proteins that play critical roles in signal transduction and cytoskeletal regulation, as well as in cell proliferation and apoptosis. We have been interested in studying the: (a) regulation of the isoprenoid pathway in the processing of cellular transformation induced by the K-ras oncogene, (b) role of the isoprenoid pathway in the control of cell proliferation and apoptosis, (c) role of the isoprenoid pathway in the control of cytoskeleton organization and (d) control of Rab expression by the isoprenoid pathway.     

Increased p21(ras) activity in human fibroblasts transduced with survivin enhances cell proliferation

Survivin is critically involved in mitosis and when overexpressed enhances the activity of the Aurora B kinase, a serine-threonine kinase belonging to the family of oncogenic Aurora/IpI1p-related kinases. Both proteins interact with Ras GTPase-activating protein suggesting an impact on the Ras pathway. This study aimed at defining the role of survivin in proliferation and potential transformation of cells. When survivin was overexpressed in normal human lung fibroblasts, the characteristic track lanes of fibroblasts were disturbed and the rate of cell proliferation was increased. An enhanced level of p21(ras) mRNA and protein expression and concomitant rise in levels of activated p21(ras) were observed. Despite increased proliferation cell survival remained dependent on serum and cells were not able to form colonies in soft agar assays. These data suggest that overexpression of survivin increases cell growth but, despite the increase in active p21(ras), is not sufficient to transform primary cells. Yet, in addition to its anti-apoptotic function it might contribute to the accelerated growth of tumour cells by increasing p21(ras) activity.     

Overexpressed galectin-3 in pancreatic cancer induces cell proliferation and invasion by binding ras and activating ras signaling

Pancreatic cancer (PDAC) is a lethal disease with a five-year survival of 3-5%. Mutations in K-Ras are found in nearly all cases, but K-Ras mutations alone are not sufficient for the development of PDAC. Additional factors contribute to activation of Ras signaling and lead to tumor formation. Galectin-3 (Gal-3), a multifunctional β-galactoside-binding protein, is highly expressed in PDAC. We therefore investigated the functional role of Gal-3 in pancreatic cancer progression and its relationship to Ras signaling. Expression of Gal-3 was determined by immunohistochemistry, Q-PCR and immunoblot. Functional studies were performed using pancreatic cell lines genetically engineered to express high or low levels of Gal-3. Ras activity was examined by Raf pull-down assays. Co-immunoprecipitation and immunofluorescence were used to assess protein-protein interactions. In this study, we demonstrate that Gal-3 was highly up-regulated in human tumors and in a mutant K-Ras mouse model of PDAC. Down-regulation of Gal-3 by lentivirus shRNA decreased PDAC cell proliferation and invasion in vitro and reduced tumor volume and size in an orthotopic mouse model. Gal-3 bound Ras and maintained Ras activity; down-regulation of Gal-3 decreased Ras activity as well as Ras down-stream signaling including phosphorylation of ERK and AKT and Ral A activity. Transfection of Gal-3 cDNA into PDAC cells with low-level Gal-3 augmented Ras activity and its down-stream signaling. These results suggest that Gal-3 contributes to pancreatic cancer progression, in part, by binding Ras and activating Ras signaling. Gal-3 may therefore be a potential novel target for this deadly disease.     

Cellular ras gene activity is required for full neoplastic transformation by polyomavirus.

To investigate the role of ras gene activity in cellular transformation by polyomavirus, murine C3H10T1/2 cells were rendered ras deficient by transfection with an antisense ras gene construct. Ras deficiency resulted in a partial suppression of the polyomavirus-induced transformed phenotype. The production of viral middle T antigen and its association with pp60c-src, increased membrane-associated protein kinase C activity, and morphological transformation were unaffected by the downregulation of c-ras gene expression. On the other hand, stimulated proliferation, focus formation on confluent monolayers of normal cells, and colony formation in soft agar were all greatly reduced in cells containing reduced p21ras levels. It is concluded that ras gene activity is needed for full cell transformation by polyomavirus.     

Cellular selenoproteins and the effects of selenite on cell proliferation

The incorporation of radioactive selenium into cellular proteins and the effect of selenite on proliferation were examined in human (HeLa, HT-29, and IMR-90) and mouse (3T3 and CMT-93) cell lines. Proteins incorporating selenium were detected by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Major polypeptide subunits at 60, 23, 21, 19, and 16 kD were detected in the two tumorigenic and one normal human cell lines. The 23 kD polypeptide migrated to the same position on the gel as the major subunit of human erythrocyte glutathione peroxidase. In the mouse cells, the 60 kD polypeptide was almost entirely absent; four other major selenoproteins were detected, with molecular weights similar to those in the human cells. In both mouse and human cells, the same pattern of selenoproteins was observed irrespective of whether the cells were grown in medium containing 10% fetal bovine serum or in defined medium supplemented with 0.1 or 1 microM selenite, or with 1% serum. The effect of selenite on proliferation of HeLa, HT-29, and CMT-93 cells in medium supplemented with 10% fetal bovine serum and in serum-free medium was examined. At concentrations up to about 1 microM, selenite stimulated proliferation of the human cells slightly in serum-free medium but not in serum-supplemented medium. At concentrations of about 5 microM and higher selenite significantly inhibited proliferation of all cells in both types of media. In CMT-93 cells, this inhibition was greater in serum-free medium, but there were no significant differences in this regard in the human cells. These results demonstrate that selenium is stably incorporated into several polypeptides in human and mouse cells, that there are no significant differences in this regard among several cell lines, and slight differences between human and mouse cells. They further confirm that selenium can have a slight stimulatory effect on cell growth, and a much larger inhibitory effect, depending on its concentration.     

肿瘤细胞恶性增殖和细胞周期调控改变的分子机制

真核细胞通过复杂的细胞周期调控系统控制细胞的分裂,从而维持有机体的正常代谢和增殖.细胞周期的调控是由一系列重要的信号分子和周期蛋白家族来完成的,这些调节因子发生突变或者表达水平发生改变,将导致细胞周期调控的改变,使细胞增殖能力增强、分化减弱,丧失细胞原有的功能,最终发展成肿瘤细胞.因此细胞周期及其相关调控蛋白和信号机制成为抗肿瘤研究的热点.     

Cellular proliferation and telomerase activity in CHRF-288-11 cells

Telomerase activity is detected in many immortalized cell lines. Recent studies suggest that terminal differentiation of some of these cell lines is associated with a reduction in telomerase activity. However, the question remains whether the reduction in telomerase activity results from terminal differentiation or from cessation of cellular proliferation. This was explored in the megakaryocytic cell line CHRF-288-11. Cells were treated with phorbol 12-myristate 13-acetate (PMA), which induces terminal differentiation of CHRF-288-11 cells, EGTA, serum depletion, and okadaic acid. All treatments resulted in cessation of proliferation. Except for okadaic acid, these treatments also induced inhibition of telomerase within 7 days. Restoring the original growth conditions of cells treated with PMA, EGTA and serum depletion resulted in the reversal of telomerase inhibition and an acceleration of proliferation. Apparent inhibition of telomerase was observed to follow the cessation of proliferation, whereas enhanced telomerase activity was noted to precede acceleration in proliferation. Thus, telomerase activity usually reflects the proliferative status rather than the differentiated status of CHRF-288-11 cells.     

Cellular proliferation and hypusine synthesis

Hypusine (N(-)-(4-amino-2-hydroxybutyl) lysine), a spermidine-dependent post-translational protein modification, is synthesized by various mammalian cells in culture. Experiments described in this paper demonstrated a relationship between rates of cellular growth and the synthesis of hypusine. Cells that divide at fast rates have a high rate of hypusine synthesis. In kinetic experiments, a positive relationship is evident between the rates of protein, DNA and hypusine synthesis. Cells seeded at high density, growing non-exponentially, synthesized less hypusine than logarithmically growing cells seeded at low density. Slowing the growth rate of cells by modification of the external milieu also results in a decreased rate of hypusine synthesis. These results provide additional evidence of the association of hypusine with cell proliferation in cultured cell lines and suggest a possible role for this unusual post-translational modification in the complex macromolecular events leading to cellular growth.     

Cellular energetics as a target for tumor cell elimination

Investigation of cancer cell metabolism has revealed variability of the metabolic profiles among different types of tumors. According to the most classical model of cancer bioenergetics, malignant cells primarily use glycolysis as the major metabolic pathway and produce large quantities of lactate with suppressed oxidative phosphorylation even in the presence of ample oxygen. This is referred to as aerobic glycolysis, or the Warburg effect. However, a growing number of recent studies provide evidence that not all cancer cells depend on glycolysis, and, moreover, oxidative phosphorylation is essential for tumorigenesis. Thus, it is necessary to consider distinctive patterns of cancer metabolism in each specific case. Chemoresistance of cancer cells is associated with decreased sensitivity to different types of antitumor agents. Stimulation of apoptosis is a major strategy for elimination of cancer cells, and therefore activation of mitochondrial functions with direct impact on mitochondria to destabilize them appears to be an important approach to the induction of cell death. Consequently, the design of combination therapies using acclaimed cytotoxic agents directed to induction of apoptosis and metabolic agents affecting cancer cell bioenergetics are prospective strategies for antineoplastic therapy.     

Capicua regulates cell proliferation downstream of the receptor tyrosine kinase/ras signaling pathway

Signaling via the receptor tyrosine kinase (RTK)/Ras pathway promotes tissue growth during organismal development and is increased in many cancers [1]. It is still not understood precisely how this pathway promotes cell growth (mass accumulation). In addition, the RTK/Ras pathway also functions in cell survival, cell-fate specification, terminal differentiation, and progression through mitosis [2-7]. An important question is how the same canonical pathway can elicit strikingly different responses in different cell types. Here, we show that the HMG-box protein Capicua (Cic) restricts cell growth in Drosophila imaginal discs, and its levels are, in turn, downregulated by Ras signaling. Moreover, unlike normal cells, the growth of cic mutant cells is undiminished in the complete absence of a Ras signal. In addition to a general role in growth regulation, the importance of cic in regulating cell-fate determination downstream of Ras appears to vary from tissue to tissue. In the developing eye, the analysis of cic mutants shows that the functions of Ras in regulating growth and cell-fate determination are separable. Thus, the DNA-binding protein Cic is a key downstream component in the pathway by which Ras regulates growth in imaginal discs.     

Human tumor cell proliferation evaluated using manganese-enhanced MRI

Background

Tumor cell proliferation can depend on calcium entry across the cell membrane. As a first step toward the development of a non-invasive test of the extent of tumor cell proliferation in vivo, we tested the hypothesis that tumor cell uptake of a calcium surrogate, Mn²⁺ [measured with manganese-enhanced MRI (MEMRI)], is linked to proliferation rate in vitro.

Methodology/Principal Findings

Proliferation rates were determined in vitro in three different human tumor cell lines: C918 and OCM-1 human uveal melanomas and PC-3 prostate carcinoma. Cells growing at different average proliferation rates were exposed to 1 mM MnCl₂ for one hour and then thoroughly washed. MEMRI R₁ values (longitudinal relaxation rates), which have a positive linear relationship with Mn²⁺ concentration, were then determined from cell pellets. Cell cycle distributions were determined using propidium iodide staining and flow cytometry. All three lines showed Mn²⁺-induced increases in R₁ compared to cells not exposed to Mn²⁺. C918 and PC-3 cells each showed a significant, positive correlation between MEMRI R₁ values and proliferation rate (p≤0.005), while OCM-1 cells showed no significant correlation. Preliminary, general modeling of these positive relationships suggested that pellet R₁ for the PC-3 cells, but not for the C918 cells, could be adequately described by simply accounting for changes in the distribution of the cell cycle-dependent subpopulations in the pellet.

Conclusions/Significance

These data clearly demonstrate the tumor-cell dependent nature of the relationship between proliferation and calcium influx, and underscore the usefulness of MEMRI as a non-invasive method for investigating this link. MEMRI is applicable to study tumors in vivo, and the present results raise the possibility of evaluating proliferation parameters of some tumor types in vivo using MEMRI.     

N-linked oligosaccharide processing and autocrine stimulation of tumor cell proliferation

Somatic mutations which impair complex-type N-linked oligosaccharide processing and chemical inhibitors of processing have been shown to reduce metastatic potential in several experimental tumor models. In this report, we demonstrate that glycosylation mutants of the metastatic MDAY-D2 tumor cell line with either truncated glycans lacking sialic acid and galactose or a mutant with less branched N-linked oligosaccharides grow more slowly in serum-free medium (SFM) than do MDAY-D2 cells. In medium containing fetal calf serum, growth rates of the cell lines were similar. A revertant of the former mutation showed a return to a more rapid growth rate in SFM. The N-linked processing inhibitor swainsonine also reduced cell growth rate in SFM but not in serum-containing medium. One of five randomly selected clones of the MDAY-D2 tumor cell line showed a slower growth rate in SFM and also showed decreased expression of branched N-linked oligosaccharides. These observations suggest that in MDAY-D2 cells, optimal factor-independent stimulation is dependent upon expression of branched complex-type N-linked oligosaccharides. The growth rate of MDAY-D2 cells in SFM was dependent on the initial seeding density of the cultures, and medium conditioned by the cells accelerated the growth of low-density cultures, suggesting that the cells respond to an autocrine factor. Culture supernatants conditioned by mutant and wild-type cells had similar levels of growth-stimulating activity. However, both mutants and swainsonine-treated cells were less responsive to this growth-stimulating activity. The growth rates of the MDAY-D2 tumor cell lines in vivo as subcutaneous tumors correlated with their relative growth rates in SFM in vitro. The results suggest that branched complex-type N-linked oligosaccharides commonly expressed in malignant cells are required for optimal autocrine-dependent growth in vitro and may be a significant factor in tumor progression in vivo.