Altered kinase C function in transformed BALB/c3T3 cells

Comparative studies of kinase C function were performed in an untransformed (A31) and the benzo[a]pyrene (BPA31), dimethylbenz[a]anthracene (DA31), and Kirsten sarcoma virus (KA31) transformed BALB/c 3T3 mouse fibroblast cell lines. The 80-kDa kinase C dependent phosphoprotein (pp80), an in vivo marker of kinase C activity, was markedly decreased in the transformed cells although the amount of the 80-kDa substrate protein in the BPA31 cells was similar to that in the untransformed A31 cells. Total cell lysate kinase C levels were lower in the transformed cells but this difference could not account for the reduced pp80 phosphorylation. Increased affinity of kinase C for the membrane fraction in the BPA31 cells may account for decreased phosphorylation of pp80.     

CONTACT-INHIBITED REVERTANT CELL LINES ISOLATED FROM SV40-TRANSFORMED CELLS : IV. Microfilament Distribution and Cell Shape in Untransformed, Transformed, and Revertant Balb/c 3T3 Cells

A comparison is made of the ultrastructure of the cell periphery in three cloned cell lines: untransformed Balb/c 3T3 cells, SV40-transformed Balb/c 3T3 cells, and revertant cells obtained from the transformed cell line by a selection technique utilizing concanavalin A. Both thin-section and surface replication techniques are used for in situ examination of the cell lines. Microfilaments, 70 Å in diameter (called alpha filaments), are abundant in untransformed and revertant cell lines, particularly in the anterior expansions of the cells, which tend to have many microvilli and small pseudopodia. Alpha filaments are diminished in the anterior expansions of transformed cells, which contain large blunt pseudopodia and relatively few microvilli. Surface replicas confirm the impression gained from thin sections that transformed cells have a greater proportion of their cell surface involved in bulging pseudopodia than either untransformed or revertant cells. Since alpha filaments are shown to bind heavy meromyosin and are similar to F-actin, these filaments are thought to be important in cell motility. These observations suggest that a close relationship exists between decreased alpha filaments, bulging pseudopodia, and loss of contact inhibition of movement in transformed cells.     

Dose fractionation effects in plateau-phase cultures of C3H 10T1/2 cells and their transformed counterparts

A comparison of gamma-ray dose fractionation effects was made using plateau-phase cultures of C3H 10T1/2 cells and their transformed counterparts in an attempt to simulate basically similar populations of cells that differ primarily in their turnover rates. The status of cell populations with respect to their turnover rates may be an important factor influencing dose fractionation effects in early- and late-responding tissues. In this cell culture system, the rate of cell turnover was approximately three times higher for the plateau-phase transformed cultures. While the single acute dose survival curves for log-phase cells were indistinguishable, there were significant differences between the survival curves for plateau-phase cultures of the two cell types. These differences were qualitatively similar to the differences recently postulated for the survival of target cells governing early and late tissue responses. Both cell lines had a similar capacity for repair of sublethal damage, but untransformed cells had a much greater capacity to repair potentially lethal damage in plateau phase. Further, untransformed plateau-phase cultures were much more sensitive to a radiation-induced G1 (or G0 to G1) delay than transformed cultures. Multifraction survival curves were determined for both cell lines for doses per fraction ranging from 9.0 to 0.8 Gy, and from these isoeffect curves of log total dose versus dose per fraction were derived. The isoeffect curve for the slowly cycling, untransformed cells was found to be appreciably steeper than that for the more rapidly cycling transformed cells, a finding consistent with previously reported differences in dose fractionation isoeffect curves for early- and late-responding tissues in vivo.     

Quantitative biochemical analysis of microtubule content in normal and transformed 3T3 cells

Microtubules in normal and transformed BALB 3T3 cells were preserved in a stabilizing medium and measured by a [3H]colchicine-binding tubulin assay, and compared to total cellular tubulin measured under nonstabilizing conditions. Essentially no change in tubulin or microtubule content was seen with changes in cell density or with changes in cellular morphology at various stages of growth of normal or transformed cells or induced by dibutyryl cAMP treatment of transformed cells. Of five cell lines transformed by a variety of agents, four had a significantly higher total tubulin content than untransformed 3T3 cells and all of them had an increased microtubule content. None of the transformed lines had a lower fraction of tubulin recoverable as sedimentable microtubules compared to untransformed cells, and in three of them this fraction was significantly higher. These results establish that microtubules are present in transformed cells to at least the extent (if not greater) than in normal cells but that there are variations in the total amount of tubulin and microtubules as well as the fraction of the total tubulin present as microtubules which are not strictly correlated with transformation or cell morphology.     

Changes in the uptake of 2-deoxy-D-glucose in BALB-3T3 cells chemically transformed in culture

Balb/3T3 cells transformed in culture by chemical carcinogens were shown to multiply in a medium supplemented with 2% calf serum or with 10% agamma new-born calf serum. The cell lines that multiply well in medium supplemented with 10% agamma serum produced a higher incidence of tumors in X-irradiated weanling mice than the lines that multiply poorly. The difference in 2-deoxy-D-glucose uptake into exponentially growing transformed and un-transformed cells was 50–100%. In crowded cultures untransformed Balb/3T3 cells ceased taking up the sugar, while chemically transformed cells continued at the same rate even at high cell densities; thus, the difference became greater in crowded cultures. When the serum concentration in the media was reduced from 10% to 2%, untransformed Balb/3T3 cells took up the sugar at a reduced rate, while chemically transformed cells were only slightly affected; agamma new born calf serum supplemented medium had no effect on sugar uptake in any of the cells. When the serum concentration was changed from 2% to 10%, untransformed cells increased sugar uptake followed by cell division. The immediacy (within 15 min) of the response in the sugar uptake to 10% serum concentration suggested that the increased uptake rate and the consequent higher concentration of the sugar (D-glucose in normal situation) within Balb/3T3 cells triggered the cell cycle. Chemical carcinogens appear to alter permanently the uptake mechanism for a key nutrient.     

Altered levels and protein kinase C-mediated phosphorylation of substrates in normal and transformed mouse lung epithelial cells.

Protein phosphorylation and protein kinase C (PKC) levels were analyzed in intact cultures of spontaneously transformed, chemically transformed, and untransformed mouse pulmonary epithelial cell lines. It was found that although the transformed cell lines contained about 80% less protein kinase C, measured as total enzyme activity or binding of [3H]phorbol ester, phosphorylation events after phorbol ester treatment could still be easily detected. A commonly described Mr 80-kDa protein kinase C substrate (p80, 80 K, MARKS) was identified using 2D-PAGE, following phosphorylation in intact cells, and found to have reduced availability for phosphorylation in the transformed cell lines C4SE9, C1SA5 and NULB5 in comparison to the untransformed C4E10 and C1C10. Available levels of p80 were further analyzed in heat-denatured extracts from all cell lines using partially purified bovine brain PKC and correlated well with changes seen in intact cells. It was also noted that all transformed cell lines contained large amounts of a family of phosphoproteins of Mr 55-65 kDa, that could not be detected in the untransformed cell lines and whose phosphorylation state was increased by protein kinase C activation. This protein was found to be located in the nucleus. Hence, spontaneously and chemically transformed mouse pulmonary epithelial cells exhibit reduced levels of PKC, along with an altered pattern of PKC-mediated phosphorylation.     

Transformation by v-H-ras does not restore proliferation of a set of temperature-sensitive cell-cycle mutants of rat 3Y1 fibroblasts

Three temperature-sensitive cell-cycle mutants of rat 3Y1 fibroblasts (3Y1tsD123, 3Y1tsG125, and 3Y1tsH203, each belonging to distinct complementation groups) were transformed with plasmid DNA carrying Harvey murine sarcoma virus cDNA. The criteria for transformation were increase in saturation cell density, capability to clone in soft agar, and alteration in the cellular morphology. At 39.8 degrees C (restrictive temperature of the parental cell lines), all the transformed sublines of each mutant ceased to proliferate and were arrested reversibly in the G1 phase of the cell cycle like the parental lines. At both 39.8 degrees C and 33.8 degrees C (permissive temperature for the parental lines), all the untransformed parental lines synthesized p21ras at low rate. At 33.8 degrees C, all the transformed sublines synthesized p21ras at much higher rate and expressed the morphological phenotype characteristic to v-H-ras-induced transformation. At 39.8 degrees C, the rate of p21ras synthesis was not changed in the transformed sublines of 3Y1tsD123 and 3Y1tsG125, and the morphology of transformed phenotype also remained intact. In the transformed subline of 3Y1tsH203, the rate of p21ras synthesis was lowered at 39.8 degrees C to that seen in the untransformed parental line, and the transformed phenotype in morphology disappeared. In all of the transformed sublines, the amount of v-H-ras mRNA markedly expressed at both 33.8 degrees C and 39.8 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin activation of protein kinase C: a reassessment

Although insulin is known to activate several protein serine/threonine protein kinases, its ability to activate protein kinase C remains controversial. We reinvestigated this question, taking advantage of several technical advances such as the development of fibroblast cell lines that overexpress normal human insulin receptors, and the development of antibodies to and expression vectors for the myristoylated, alanine-rich C kinase substrate (MARCKS) protein, a major cellular substrate for protein kinase C. In HIR 3.5 cells, a mouse 3T3 cell derivative that expresses about 6 x 10(6) human insulin receptors/cell, insulin (70 nM for 10 min) stimulated phosphorylation of the MARCKS protein by approximately 2-fold (p less than 0.005). This phosphorylation was not further increased by different times of insulin exposure, different insulin concentrations, or longer periods of serum deprivation. The insulin stimulation represented about 14% of the response to phorbol 12-myristate 13-acetate and about 17% of the response to 10% fetal calf serum. No significant stimulation of MARCKS protein phosphorylation was seen in four other insulin-sensitive cell lines, in which insulin is known to activate other protein serine/threonine kinases: HIRC-B, BC3H-1, 3T3-L1 adipocytes, and H35 rat hepatoma cells made to stably express the MARCKS protein. In these four cell lines, serum and/or phorbol 12-myristate 13-acetate exerted a large stimulatory effect on MARCKS protein phosphorylation. We conclude that insulin may activate protein kinase C to a minor extent in certain cell types that vastly overexpress insulin receptors; however, we believe that this effect of insulin is unlikely to be of physiological importance.     

Differential Expression of MARCKS and Other Calmodulin-Binding Protein Kinase C Substrates in Cultured Neuroblastoma and Glioma Cells

Abstract: Expression of the protein kinase C substrate MARCKS and other heat-stable myristoylated proteins have been studied in four cultured neural cell lines. Amounts of MARCKS protein, measured by [³H]myristate labeling and western blotting, were severalfold higher in rat C6 glioma and human HTB-11 (SK-N-SH) neuroblastoma cells than in HTB-10 (SK-N-MC) or mouse N1E-115 neuroblastoma cells. Higher levels of MARCKS mRNA were also detected in the former cell lines by S1 nuclease protection assay. At least two additional ³H-myristoylated proteins of 50 and 40–45 kDa were observed in cell extracts heated to >80°C or treated with perchloric acid. The 50-kDa protein, which bound to calmodulin in the presence of Ca²⁺, was more prominent in cells (N1E-115 and HTB-10) with less MARCKS, whereas neuromodulin (GAP-43) was detected in N1E-115 and HTB-11 cells only. Heating resulted in a fourfold increase in the detection of MARCKS by western blotting; this was not paralleled by a similar increase in [³H]myristate-labeled MARCKS and may be due to a conformational change affecting the C-terminal epitope or enhanced retention of the protein on nitrocellulose. Addition of β-12- O -tetradecanoylphorbol 13-acetate resulted in three- to fourfold increased phosphorylation of MARCKS in HTB-11 cells, with little increase noted in HTB-10 cells. These results indicate that MARCKS, neuromodulin, and other calmodulin-binding protein kinase C substrates exhibit distinct levels of expression in cultured neurotumor cell lines. Of these proteins, only MARCKS appears to be correlated with phorbol ester stimulation of phosphatidylcholine turnover in these cells.     

Coupled Activation of Mechanosensitive and Calcium-Dependent Potassium Channels in 3T3 and 3T3-SV40 Cells

Using the patch–clamp method, mechanosensitive regulation of ion channels was studied in cultivated 3T3 and 3T3-SV40 fibroblasts. The activity of mechanosensitive cation channels with a conductivity 25 pS in response to plasma-membrane stretching was observed in both cell lines. Despite obvious differences in the actin network in normal and transformed cells, the threshold values of the stimulus required for the channel activation were close and were approximately 55 mm Hg. The frequency of channels was significantly higher in transformed 3T3-SV40 fibroblasts than in their untransformed 3T3 analogs. Coupled activation of mechanosensitive calcium-permeable channels and potassium calcium-controlled channels was found in both cell lines. The analysis of flows through single channels allows to detect functional interaction of different channels: stretch-induced local calcium entry activates potassium channels that do not have their own mechanosensitivity. The results of a comparative study show that there is a fundamental similarity between the ion mechanisms of cellular mechanotransduction in normal and transformed fibroblasts. The quantitative differences, first of all, concern the level of functional activity of mechanosensitive channels that provide the development of the local calcium signal in the near-membrane cell region.     

Selective effects of trypsinization on established and tumour-derived mouse 3T3 cells.

Explanted tumour cells are much more sensitive to the deleterious effects of routine trypsinization than are the parent 3T3 or SV40 transformed established cell lines. This differential sensitivity causes the disappearance of tumour-derived cells when grown in co-culture with untransformed 3T3 cells and accounts in some tumor explants for the emergence of trypsin-resistant varient cells which have lost tumour-specific properties.     

Biochemical Studies on Bovine Adenovirus Type 3 IV. Transformation by Viral DNA and DNA Fragments

By the calcium technique, intact DNA of bovine adenovirus type 3 (BAV3) was found to transform A31 cells, a clone of BALB/3T3. Transforming activity was resistant to RNase and Pronase but sensitive to DNase. The efficiency of transformation was approximately 5 to 10 foci per μg of DNA. Attempts were also made to test for transforming activity of BAV3 DNA fragments prepared with restriction endonucleases EcoRI and HindIII. The activity was found to associate exclusively with the EcoRI D fragment mapped in the region of 3.6 and 19.7 units (molecular weight, 3.9 × 10⁶). No transformation could be obtained with three HindIII fragments, J, E, and B, located at the left-hand end of the BAV3 genome. However, the enzymatic joining of J and E fragments (0 to 11.9 map units) with a ligase restored the transforming activity. These results suggest that all the genetic information of BAV3 required for transformation is located in the region between 3.6 and 11.9 units on the viral genome. Some properties of A31 cells transformed by BAV3 DNA EcoRI D fragment (TrD) and the ligated DNA of HindIII J and E fragments (TrJE), as well as those transformed by whole BAV3 DNA (Tr), were examined. As compared to untransformed A31 cells, all the transformed cell lines tested showed rapid growth, high saturation densities, and anchorage-independent growth. Moreover, they contained BAV3-specific T antigen and induced tumors in adult nude and BALB/c mice. These properties of Tr, TrD, and TrJE lines were similar to those of BAV3-transformed cells.     

Density Dependent Change of Myristoylated Proteins in C3H10T1/2 Fibroblasts and Their Transformants

We have examined the pattern of protein myristoylation in C3H10T1/2 fibroblasts during cell growth. During the growing phase of 10T1/2 cells, several proteins were radiolabelled with [³H]myristate, and among them proteins with molecular masses of 22, 35, a doublet of 42–45 and 67 kDa were labelled predominantly. The extent of myristoylation in each of these proteins changed with cell density. The amount of radioactivity incorporated into the 22 kDa protein in 10T1/2 cells decreased with increasing cell density and remained at a low level during the stationary phase. In contrast, the incorporation into the 67 kDa protein increased parallel to cell density. The density-dependent change of myristoylation was not observed in any of the transformants of 10T1/2 cells thus far examined. The 67 kDa protein was identified as MARCKS (myristoylated alanine-rich C kinase substrate) by immunoprecipitation with an anti-MARCKS antibody. By Western blot analysis, we found that the amount of MARCKS in 10T1/2 cells increased significantly analogous with cell density. Therefore, it is possible that MARCKS and the 22 kDa protein play a role in contact-mediated cell signalling in 10T1/2 cells, but the mechanism is lost in transformed cells. © 1997 John Wiley & Sons, Ltd.     

Endogenous butyrylcholinesterase in SV40 transformed cell lines: COS-1, COS-7, MRC-5 SV40, and WI-38 VA13

Summary Comparison of proteins expressed by SV40 transformed cell lines and untransformed cell lines is of interest because SV40 transformed cells are immortal, whereas untransformed cells senesce after about 50 doublings. In MRC-5 SV40 cells, only seven proteins have previously been reported to shift from undetectable to detectable after transformation by SV40 virus. We report that butyrylcholinesterase is an 8th protein in this category. Butyrylcholinesterase activity in transformed MRC-5 SV40 cells increased at least 150-fold over its undetectable level in MRC-5 parental cells. Other SV40 transformed cell lines, including COS-1, COS-7, and WI-38 VA13, also expressed endogenous butyrylcholinesterase, whereas the parental, untransformed cell lines, CV-1 and WI-38, had no detectable butyrylcholinesterase activity or mRNA. Infection of CV-1 cells by SV40 virus did not result in expression of butyrylcholinesterase, showing that the butyrylcholinesterase promoter was not activated by the large T antigen of SV40. We conclude that butyrylcholinesterase expression resulted from events related to cell immortalization and did not result from activation by the large T antigen.     

Metabolic responses of Taxus media transformed cell cultures to the addition of methyl jasmonate

Dedifferentiated Taxus media cell cultures presenting the same genetic characteristics as the parent culture were established from transformed roots. Two transformed cell lines were studied: Rol C, carrying the T‐DNA of A. rhizogenes 9,402 and TXS, carrying both the T‐DNA of A. rhizogenes and the txs transgene of T. baccata under the control of the 35S CaMV promoter. In the second part of a previously optimized two‐stage system, the transformed cell lines were cultured in a production medium supplemented with the elicitor methyl jasmonate. Taxane production in the transformed cultures was compared with an untransformed T. media cell line cultured in the same conditions. The highest taxane production was observed in the TXS cell line when cultured in the optimized production medium with methyl jasmonate, being 265% greater than in the untransformed control and 170% greater than in the Rol C cell line. However, txs expression and the activity of the enzyme taxadiene synthase in the TXS cells were lower than in the line carrying only the rol genes (Rol C). It is also noteworthy that the taxane production as well as the txs gene expression and TXS activity in all the cell lines, both transformed and untransformed, were clearly dependent on the elicitor action. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Properties of a sarcoma-growth-factor-like peptide from cells transformed by a temperature-sensitive sarcoma virus

Serum-free conditioned media was collected from three sarcoma virus-transformed cell lines and an untransformed cell line. All three virally transformed lines produced and released growth factors into their serum-free media. The major activity in all cases, whether the cells were transformed by Moloney sarcoma virus (MSV) or Kirsten sarcoma virus (KiSV), or whether they were mouse or rat, was a sarcoma-growth-factor (SGF)-like activity with an apparent molecular weight of 10,000. The SGF-like pools from a Moloney sarcoma virus-transformed mouse 3T3 cell and a Kirsten sarcoma virus-transformed NRK cell were further purified by carboxymethyl cellulose chromatography. The elution profiles of these peptides were very similar. The serum-free conditioned media from the untransformed cells showed no detectable growth stimulating activity. The temperature sensitivity of an SGF-like growth factor from the supernate of a NRK cell transformed by a wild-type Kirsten sarcoma virus (KiSV) was compared with that of the SGF-like activity from the supernates of a NRK cell transformed by a ts-mutant of KiSV that is temperature sensitive with respect to transformation (ts-371 Cl 5). Neither the cells transformed by the wild-type sarcoma virus nor those transformed by the temperature sensitive virus released a SGF-like activity that was temperature sensitive under the conditions of the assays.     

Dissociation of Phosphorylation and Translocation of a Myristoylated Protein Kinase C Substrate (MARCKS Protein) in C6 Glioma and N1E-115 Neuroblastoma Cells

Abstract: An 80-kDa protein labeled with [³H]myristic acid in C6 glioma and N1E-115 neuroblastoma cells has been identified as the myristoylated alanine-rich C kinase substrate (MARCKS protein) on the basis of its calmodulin-binding, acidic nature, heat stability, and immunochemical properties. When C6 cells preincubated with [³H]myristate were treated with 200 n M 4β-12- O -tetradecanoylphorbol 13-acetate (β-TPA), labeled MARCKS was rapidly increased in the soluble digitonin fraction (maximal, fivefold at 10 min) with a concomitant decrease in the Triton X-100–soluble membrane fraction. However, phosphorylation of this protein was increased in the presence of β-TPA to a similar extent in both fractions (maximal, fourfold at 30 min). In contrast, β-TPA–stimulated phosphorylation of MARCKS in N1E-115 cells was confined to the membrane fraction only and no change in the distribution of the myristoylated protein was noted relative to α-TPA controls. These results indicate that although phosphorylation of MARCKS by protein kinase C occurs in both cell lines, it is not directly associated with translocation from membrane to cytosol, which occurs in C6 cells only. The cell-specific translocation of MARCKS appears to correlate with previously demonstrated differential effects of phorbol esters on stimulation of phosphatidylcholine turnover in these two cell lines.     

Elevated prostaglandins synthetase activity in methylcholanthrene-transformed mouse BALB/3T3.

Cell lines transformed from 3T3 spontaneously, by radiation, or by treatment with chemical carcinogens, polyoma and SV40 virus produce up to 5 times more prostaglandins than their untransformed parent line. Several aspects of prostaglandin biosynthesis by MC5-5 and 3T3 were compared. When stimulated by serum, bradykinin, or thrombin, MC5-5 cells labeled with radioactive arachidonic acid in their cellular lipids, these higher levels were shown not to be due to increased availability of the prostaglandin precursor, arachidonic acid. Prostaglandin synthetase activity in microsomal fractions prepared from MC5-5 was 6 times higher than that of microsomes of untransformed cells. The increased prostaglandin levels produced by transformed cells therefore appear to be the result of elevated prostaglandin synthetase activity.     

Elevated prostaglandin synthetase activity in methylcholanthrene-transformed mouse BALB/3T3

Cell lines transformed from 3T3 spontaneously, by radiation, or by treatment with chemical carcinogens, polyoma and SV40 virus produce up to 5 times more prostaglandins than their untransformed parent line. Several aspects of prostaglandin biosynthesis by MC5-5 and 3T3 were compared. When stimulated by serum, bradykinin, or thrombin, MC5-5 produced 2-to 5-fold more prostaglandins than 3T3. With the use of cells labeled with radioactive arachidonic acid in their cellular lipids, these higher levels were shown not to be due to increased availability of the prostaglandin precursor, arachidonic acid. Prostaglandin synthetase activity in microsomal fractions prepared from MC5-5 was 6 times higher than that of microsomes of untransformed cells. The increased prostaglandin levels produced by transformed cells therefore appear to be the result of elevated prostaglandin synthetase activity.