Mouse Ehrlich Ascites Tumor Cells Research Articles

Combined treatment with dissolved hydrogen molecule and platinum nanocolloid exerts carcinostatic/carcinocidal effects by increasing hydrogen peroxide generation and cell death in the human gastric cancer cell line NUGC-4

Many studies have shown that redox regulation is an effective therapeutic strategy for different types of cancer. We have previously demonstrated that combined treatment with dissolved hydrogen molecule (H2) and platinum nanocolloid (Pt-nc) has carcinostatic effects and that increased intracellular reactive oxygen species (ROS) levels were closely associated with carcinostatic effects in Ehrlich mouse ascites tumor cells. However, it is unknown whether combined treatment-induced ROS generation can occur in human cancer cells. Therefore, this study aimed to examine the carcinostatic effect of the combined treatment in human cells and investigate the relationship between treatment efficacy and ROS generation. H2 and Pt-nc treatment could exert cytostatic action by inhibiting the growth of human promyelocytic leukemia HL60 and human gastric adenocarcinoma-derived NUGC-4 cells; however, no effect was observed in normal human embryo fibroblast OUMS-36 cells by the temporary exposure. These findings indicate that combined treatment with H2 and Pt-nc may act selectively in tumor cells compared with normal cells. Additionally, combined treatment with H2 and Pt-nc resulted in an approximately 200-fold increase in intracellular ROS levels compared with the control, whereas the suppressive effect of tumor cell growth was abrogated entirely by catalase treatment in NUGC-4 cells. Furthermore, combined treatment with H2 and Pt-nc induced hydrogen peroxide generation, cellular morphological changes, cell death, and a decline in DNA synthesis-positive cells. In conclusion, combined treatment with H2 and Pt-nc can induce carcinostatic/carcinocidal effects through intracellular ROS increase, morphological changes, cell death, and DNA synthesis suppression in the human tumor cell line.

Melatonin effect on the ultrastructure of Ehrlich ascites tumor cells, lifetime and histopathology in Swiss mice

AimsOne of the models used for studying cancer is the Ehrlich ascites tumor (EAT) due to its ability to grow in liquid suspension, allowing a standard number of cells to be inoculated, growth quantification and regression of tumor mass. Among the oncostatic substances, melatonin has shown effectiveness in limiting the tumor cell proliferation. However, studies have shown contradictory effects of melatonin on the EAT. This study has investigated the melatonin effect on tumor growth, time and survival percentage, ultrastructure and metastasis of EAT cells in mice submitted or not to pinealectomy. Main methodsAnimals were inoculated with 5×106cells/mL and treated or not with exogenous melatonin with doses of at 150 and 300μg/30g animal weight for 12days. Melatonin significantly reduced the abdominal circumference, volume of ascites liquid and EAT-cell viability, raising rates of time and mice survival percentage. Key findingsUltrastructurally, the melatonin treatment revealed changes in the shape of cells, the cell surface showed numerous projections, some bifurcated, cytoplasmic vacuolation, mitochondrial degeneration and nuclear fragmentation, peculiar characteristics of apoptosis. Histopathology revealed no metastasis in the liver, small intestine and large intestine in any of the animals in the experimental groups; however this process was evident in the lungs and kidneys, being inhibited by melatonin administration. SignificanceThus, we can conclude that doses of 150 and 300μg/30g of melatonin for 12 consecutive days have a very effective oncostatic and cytotoxic activity on EAT cells in mice.

Novel Glycolipoproteins from Ginseng

Ginseng has been used as a general tonic agent to invigorate human body. In the present study, we isolated novel glycolipoproteins from ginseng that activate Ca²?-activated Cl- channel (CaCC) in Xenopus oocytes and transiently increase intracellular free Ca²? concentration ([Ca²?] i ) in mouse Ehrlich ascites tumor cells. We named the active ingredients as gintonin. Gintonin exists in at least six different forms. The native molecular weight of gintonin is about 67 kDa but its apparent molecular weight is about 13 kDa, indicating that gintonin might be a pentamer. Gintonin is rich in hydrophobic amino acids. Its main carbohydrates are glucose and glucosamine. Its lipid components are linoleic, palmitic, oleic, and stearic acids. Gintonin actions were blocked by U73122, a phospholipase C inhibitor, 2-aminoethxydiphenyl borate, an inositol 1,4,5-trisphosphate receptor antagonist, or bis (o-aminophenoxy) ethane-N,N,N0,N0-tetracetic acid acetoxymethyl ester, a membrane permeable Ca²? chelator. In the present study, we for the fi rst time isolated novel gintonin and showed the signaling pathways on gintonin-mediated CaCC activations and transient increase of [Ca²?] i . Since [Ca²?] i as a second messenger plays a pivotal role in the regulation of diverse Ca²?-dependent intracellular signal pathways, gintonin-mediated regulations of [Ca²?] i might contribute to biological actions of ginseng.

Endothelin-1 Stimulates Secretion of Lipoprotein Lipase from Ehrlich Ascites Tumor Cells

Since the role of endothelin (ET)-1 in lipoprotein metabolism in tumor cells is unclear, we investigated the effect of ET-1 on the secretion of lipoprotein lipase (LPL) from mouse Ehrlich ascites tumor cells. ET-1 increased the secretion of LPL from these cells in a time-dependent manner. Two antagonists of ET-receptor type A (ET-A), namely, BQ123 and FR139317, inhibited the stimulatory effect of ET-1 on the secretion of LPL. However, an antagonist of ET-receptor type B (ET-B), BQ788, did not have any effect. Neomycin, a phospholipase C (PLC) inhibitor, and H-7, a protein kinase C (PKC) inhibitor, also suppressed the ET-1-stimulated secretion of LPL. ET-1 also increased PKC activity in tumor cells in a dose-dependent manner. These results imply that ET-1 stimulates secretion of LPL from tumor cells by stimulating the PLC-PKC signaling pathway through the ET-A receptor rather than the ET-B receptor.

Evaluation of the Antineoplastic Activity of L-rhamnose in vitro. A Comparison with 2-deoxyglucose

The effect of unsubstituted deoxyhexoses, 2-deoxy-D-glucose (2-DG) and L-fucose, on tumor cells has been reported in several papers throughout the last decades. That of a similar deoxysugar, L-rhamnose, which is synthesized in bacteria and plants but not in animal cells, has until today not been explored. In the present study, we examined the effect of L-rhamnose on DNA and protein synthesis, growth and the potential induction of apoptosis of tumor cells in vitro. Using 2-DG for comparison, we studied the effect of L-rhamnose in concentrations up to 20 (32 resp.) mmol/l on the initial velocity of the incorporation of labeled precursors of DNA and proteins in short term cultures of both mouse Ehrlich ascites tumor (EAT) and human HL-60 cells in vitro, and further, on cell proliferation and apoptosis induction in HL-60 cells. Neither cytotoxic nor cytostatic effects of L-rhamnose were observed with the exception of slightly pronounced inhibition of DNA synthesis in EAT cells. From the lacking inhibition of the protein synthesis it can be considered that L-rhamnose does not interfere with energy metabolism, at least not in a similar manner as 2-DG.

Systemic Cancer Gene Therapy Using Adeno-associated Virus Type 1 Vector Expressing MDA-7/IL24

Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL24), selectively induces apoptosis in cancer cells without harming normal cells. It also exerts immunomodulatory and antiangiogenic effects, as well as potent antitumor bystander effects, making it an ideal candidate for a new anticancer gene therapy. Here, we examined the feasibility of adeno-associated virus type 1 (AAV1) vector-mediated systemic gene therapy using mda-7/IL24. In vitro studies showed that medium conditioned by AAV1-mda7-transducedC2C12 cells induces tumor cell-specific apoptosis and inhibits angiogenesis in a human umbilical vein endothelial cell tube formation assay. To assess the in vivo effects of AAV1-mediated systemic delivery of MDA-7/IL24, we generated a subcutaneous tumor model by injecting Ehrlich ascites tumor cells into the dorsum of DDY mice. A single intravenous injection of AAV1-mda7 (2.0 x 10(11) viral genomes) significantly inhibited tumor growth. In addition, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), and immunohistochemical analyses showed significant induction of tumor-cell-specific apoptosis and reduction of microvessel formation within the tumors, and there was a significant increase in survival among the AAV1-mda7-treated mice. These results clearly demonstrate that continuous systemic delivery of MDA-7/IL24 can serve as an effective treatment for cancer. Thus, AAV1 vector-mediated systemic delivery of MDA-7/IL24 represents a potentially important new approach to anticancer therapy.

Cell content of phosphatidylinositol (4,5)bisphosphate in Ehrlich mouse ascites tumour cells in response to cell volume perturbations in anisotonic and in isosmotic media

The labelling pattern of cellular phosphoinositides (PtdInsP(n)) was studied in Ehrlich ascites cells labelled in vivo for 24 h with myo-[2-(3)H]- or l-myo-[1-(3)H]inositol and exposed to anisotonic or isosmotic volume perturbations. In parallel experiments the cell volume ([(14)C]3-OMG space) was monitored. In hypotonic media the cells initially swelled osmotically and subsequently as expected showed a regulatory volume decrease (RVD) response. Concurrently, the cell content of PtdInsP(2) showed a marked, transient decrease and the content of PtdInsP a small, transient increase. The changes in PtdInsP(2) and PtdInsP content increased progressively with the extent of hypotonicity (in the range 1.00-0.50 relative osmolarity). No evidence was found for either hydrolysis of PtdInsP(2) or formation of PtdInsP(3). In hypertonic medium (relative osmolarity 1.50), cells initially shrank osmotically and subsequently as expected showed a small regulatory volume increase (RVI) response. Concurrently, the cell content of PtdInsP(2) showed a marked increase and the content of PtdInsP a small decrease, i.e. changes in the opposite direction of those seen in hypotonic media. In isosmotic media with high (100 mm) or low (0.8 mm) K(+) concentration, cells slowly swelled or shrank due to uptake or loss of isosmotic KCl. Under these conditions, with largely unchanged intracellular ionic strength, the cell content of PtdInsP(2) and PtdInsP remained constant. Our results show that PtdInsP(2) is not volume sensitive per se, and moreover that the regulatory volume adjustments in Ehrlich ascites cells are not mediated by PtdInsP(2) hydrolysis and its subsequent production of second messengers. The simplest interpretation of the observed effects would be that PtdInsP(2) is controlled by ionic strength, probably via activation/inhibition of phosphoinositide-specific phosphatases/kinases. In Ehrlich ascites cells, as shown previously, the opposing ion channels and transporters activated during RVD and RVI, respectively, are controlled with tight negative coordination by a common cell volume 'set-point' that is shifted in anisotonic media, but unchanged during cell swelling in isosmotic high K(+) medium. We hypothesize that PtdInsP(2) might orchestrate this 'set-point' shift.

Lysophosphatidylcholine-induced taurine release in HeLa cells involves protein kinase activity

It has recently been demonstrated that exogenous addition of low concentrations (<15 μM) of lysophosphatidyl choline (LPC, palmitic acid in the sn-1 position) induces a transient increase in taurine efflux from HeLa cells in a process that seems to involve generation of reactive oxygen species (ROS) and tyrosine phosphorylation (J. Membrane Biol. 176 (2000) 175–185). We now demonstrate that LPC also induces release of taurine under isotonic conditions in mouse fibroblast (NIH/3T3) and Ehrlich ascites tumor cells. Furthermore, we show that in the case of HeLa cells addition of the calmodulin antagonist W-7 (50 μM) or the calmodulin-dependent kinase II (CaMKII) inhibitor KN-62 (10 μM) reduces the LPC-induced taurine release under isotonic conditions. Conversely, addition of a standard protein kinase C (PKC) inhibitor chelerythrine (10 μM) leads to a potentiation of the LPC-induced taurine efflux, whereas direct activation of PKC by the phorbol ester PMA has no effect. It is suggested that the putative generation of ROS following addition of LPC is modulated by calmodulin/CaMKII, and that the effect of chelerythrine is more likely related to the ROS production than to PKC inhibition.

Characterisation of a cell swelling-activated K+-selective conductance of ehrlich mouse ascites tumour cells.

The K+ and Cl- currents activated by hypotonic cell swelling were studied in Ehrlich ascites tumour cells using the whole-cell recording mode of the patch-clamp technique. Currents were measured in the absence of added intracellular Ca2+ and with strong buffering of Ca2+. K+ current activated by cell swelling was measured as outward current at the Cl- equilibrium potential (ECl) under quasi-physiological gradients. It could be abolished by replacing extracellular Na+ with K+, thereby cancelling the driving force. Replacement with other cations suggested a selectivity sequence of K+ > Rb+ > NH4 approximately Na+ approximately Li+; Cs+ appeared to be inhibitory. The current-voltage relationship of the volume-sensitive K+ current was well fitted with the Goldman-Hodgkin-Katz current equation between -130 and +20 mV with a permeability coefficient of around 10(-6) cm s(-1) with both physiological and high-K+ extracellular solutions. The class III antiarrhythmic drug clofilium blocked the volume-sensitive K+ current in a voltage-independent manner with an IC50 of 32 microM. Clofilium was also found to be a strong inhibitor of the regulatory volume decrease response of Ehrlich cells. Cell swelling-activated K+ currents of Ehrlich cells are voltage and calcium insensitive and are resistant to a range of K+ channel inhibitors. These characteristics are similar to those of the so-called background K+ channels. Noise analysis of whole-cell current was consistent with a unitary conductance of 5.5 pS for the single channels underlying the K+ current evoked by cell swelling, measured at 0 mV under a quasi-physiological K+ gradient.

Distribution of3H within purine nucleotides of Ehrlich mouse ascites tumour cells after intraabdominal injection ofmyo-[2-3H]inositol

In Ehrlich mouse ascites tumour cells, exposed intra-abdominally to [2-3H]inositol, ATP and GTP presented enough aberrant 3H-label to cause potential interference in the chromatographic analysis of inositol phosphates involved in signal transduction. After acid extraction and charcoal adsorption/desorption the nucleotides were dephosphorylated, enriched with [U-14C]adenosine, and exposed to purine-nucleoside specific enzymes. Reverse phase HPLC and radioactivity measurement demonstrated that for adenosine about 82% of total stable 3H label was in ribose and thus about 18% in adenine. For guanosine about 89% was in ribose and 11% in guanine. This aberrant 3H labelling could be avoided using [1-3H]inositol. Copyright © 2000 John Wiley & Sons, Ltd.

Distribution of 3H within purine nucleotides of Ehrlich mouse ascites tumour cells after intraabdominal injection of myo‐[2‐3H]inositol

Distribution of 3H within purine nucleotides of Ehrlich mouse ascites tumour cells after intraabdominal injection of myo‐[2‐3H]inositol

Transcriptionally active and inactive mouse beta-globin gene loci are repaired at similar rates after ultraviolet irradiation.

It has been demonstrated, by Northern blot and in situ hybridization, that the mouse erythroleukaemia cell line F4N-Sofia constitutively expresses the beta-globin genes. The recently developed quantitative assay for DNA repair has been used to study the overall repair rate in the beta-globin gene domain in this cell line after ultraviolet irradiation and to compare it with the repair rate of the same chromatin domain in mouse Ehrlich ascites tumour cells which do not express the beta-globin genes. The results showed that in both cases the 5'-end of the domain was repaired preferentially and that the repair rates in the two cell lines were very similar despite the different transcription state of the genes.

Molecular Cloning and Expression of a Mouse Brain cDNA Encoding a Novel Protein Target of Calcyclin

A protein target of mouse calcyclin, p30, which we call calcyclin-binding protein (CacyBP), was identified in mouse brain and Ehrlich ascites tumor (EAT) cells. The amino acid sequence of the CacyBP chymotryptic peptide was used to prepare synthetic oligonucleotides that served as a probe to screen the mouse brain cDNA library. A 1.4-kb positive clone was detected, isolated, and sequenced. The analyzed clone contains an open reading frame encoding a protein of a molecular mass of approximately 26 kDa. The nucleotide and predicted amino acid sequences indicate that CacyBP is a novel protein. The results obtained from northern blots show that the CacyBP gene is expressed predominantly in mouse brain and EAT cells. Using a pGEX vector the recombinant CacyBP was expressed in Escherichia coli, and its properties were analyzed. The recombinant protein interacts with calcyclin at a physiologically relevant range of Ca2+ in solution during affinity chromatography and on blots. Because CacyBP, like calcyclin, is present in the brain, the interaction of these two proteins might be involved in calcium signaling pathways in neuronal tissue.

The 58 kDa mouse selenoprotein is a BCNU-sensitive thioredoxin reductase

The flavoprotein thioredoxin reductase [EC 1.6.4.5] (NADPH+H ++thioredoxin-S 2→NADP ++thioredoxin-(SH) 2) was isolated from mouse Ehrlich ascites tumour (EAT) cells. Like the counterpart from human placenta but unlike the known thioredoxin reductases from non-vertebrate organisms, the mouse enzyme was found to contain 1 equivalent of selenium per subunit of 58 kDa. The K M values were 4.5 μM for NADPH, 480 μM for DTNB and 36 μM for Escherichia coli thioredoxin, the turnover number with DTNB being ≈40 s −1. As mouse is a standard animal model in cancer and malaria research, thioredoxin reductase and glutathione reductase [EC 1.6.4.2] from EAT cells were compared with each other. While both enzymes in their 2-electron reduced form are targets of the cytostatic drug carmustine (BCNU), no immunologic cross-reactivity between the two mouse disulfide reductases was observed.

On the Role of Calcium in the Regulatory Volume Decrease (RVD) Response in Ehrlich Mouse Ascites Tumor Cells

The putative role for Ca2+ entry and Ca2+ mobilization in the activation of the regulatory volume decrease (RVD) response has been assessed in Ehrlich cells. Following hypotonic exposure (50% osmolarity) there is: (i) no increase in cellular Ins(1,4,5)P3 content, as measured in extracts from [2-3H]myoinositol-labeled cells, a finding at variance with earlier reports from our group; (ii) no evidence of Ca2+-signaling recorded in a suspension of fura-2-loaded cells; (iii) Ca2+-signaling in only about 6% of the single, fura-2-loaded cells at 1-mm Ca2+ (1% only at 0.1-mM Ca2+ and in Ca2+-free medium), as monitored by fluorescence-ratio imaging; (iv) no effect of removing external Ca2+ upon the volume-induced K+ loss; (v) no significant inhibition of the RVD response in cells loaded with the Ca2+ chelator BAPTA when the BAPTA-loading is performed in K+ equilibrium medium; (vi) an inhibition of the swelling-induced K+ loss (about 50%) at 1-mM Ba2+, but almost no effect of charybdotoxin (100 nm) or of clotrimazole (10 microM), reported inhibitors of the K+ loss induced by Ca2+-mobilizing agonists. Thus, Ca2+signaling by Ca2+ release or Ca2+ entry appears to play no role in the activation mechanism for the RVD response in Ehrlich cells.

Characterization of chicken gizzard calcyclin and examination of its interaction with caldesmon

Using a procedure developed to purify calcyclin from mouse Ehrlich ascites tumor cells calcyclin was purified from smooth muscle of chicken gizzard. Chicken gizzard calcyclin bound to phenyl-Sepharose in a calcium dependent manner as did mouse EAT cells and rabbit lung calcyclin but appeared to be more acidic than its mammalian counterparts as revealed by ion exchange chromatography on Mono Q. Chicken gizzard calcyclin bound 45Ca2+ on nitrocellulose filters and exhibited a shift in electrophoretic mobility on urea-PAGE depending on Ca2+ concentration. Crosslinking experiments with BS3 showed that chicken gizzard calcyclin was able to form noncovalent dimers. As indicated by a decrease in maximum tryptophan fluorescence emission of caldesmon (about 14% at 1:1 molar ratio) and displacement of calmodulin from its complex with caldesmon, chicken gizzard calcyclin binds caldesmon. This binding was, however, much weaker than that of calmodulin and could not influence the interaction of caldesmon with actin. In consequence, calcyclin was unable to reverse the inhibitory effect of caldesmon on actin-activated Mg2+-ATPase activity of myosin in the presence of Ca2+.

Characterization of gangliosides from Ehrlich ascites tumour cells and their variants.

Differences in the nature of the gangliosides present in two types of Ehrlich ascites tumour (EAT) cells, the adherent and non-adherent EAT cells, were studied. Gangliosides were isolated by DEAE Sephadex column chromatography and analysed by high-performance thin-layer chromatography (HPTLC). The non-adherent EAT (na-EAT) cells which grow in the peritoneal cavity of mice were selected for growth on basement membrane and tissue culture plastic to give the adherent EAT (a-EAT) cells. na-EAT cells contained 1.57 nmol lipid-bound sialic acid per mg protein and at least 12 different gangliosides, including major gangliosides such as GM3, GM2, GM1, GD3, GD1a and GT1b. On the other hand, the ganglioside pattern of a-EAT cells differed significantly from that of na-EAT cells, both quantitatively and qualitatively. The content of lipid-bound sialic acid in a-EAT cells was only 0.24 nmol per mg of protein. The gangliosides in a-EAT cells were characterized as GD1a and trisialogangliosides and, significantly, a-EAT cells did not contain monosialogangliosides. Neutral glycolipids were isolated from both cell lines and their patterns were compared. In contrast to the gangliosides pattern, their neutral glycolipid patterns were similar. Glucosylceramide and lactosylceramide were the major components in both types of cells. In addition to na- and a-EAT cells, a-EAT cells were passaged in mice by intraperitoneal injection, giving rise to a third variant (c/m EAT cells). We analysed the gangliosides in c/m EAT cells to determine whether there was a change in the ganglioside pattern found in na-EAT cells. After repeated passage of c/m EAT cells in mice, the pattern of gangliosides shifted to that of na-EAT cells. Alterations of ganglioside composition may be associated with the growth environment of the murine peritoneal cavity; alternatively, a selection process may have occurred.

Partial purification and characterization of a protein kinase that is activated by nuclear localization signal peptides

A nuclear localization signal (NLS) is required for the transport of karyophilic proteins from the cytoplasm to the nucleus. In this study, NLS was examined in terms of its effect on diverse cellular functions such as protein phosphorylation reactions. When synthetic peptides containing the NLS of SV40 T-antigen were injected into the cytoplasm of Xenopus oocytes, and the oocytes incubated with [ 32P]phosphorus-containing medium, a 32 kDa protein was found to be preferentially phosphorylated in an NLS-dependent manner. The incubation of fractionated cytosolic extracts prepared from mouse Ehrlich ascites tumor cells with [ γ- 32P]ATP in the presence of the NLS peptides, results in the stimulation of the phosphorylation of several proteins. Similar in vitro stimulation was observed by other functional NLS peptides such as those of polyoma virus T-antigen and nucleoplasmin. Little or no stimulation, however, was detected for peptides of mutant type and reverse type NLS of SV40 T-antigen, and the C-terminal portion of lamin B. Using an in vitro assay, the phosphorylation activity was fractionated chromatographically and a fraction was obtained which contained a high level of activity. The fraction was found to contain three major proteins having molecular masses of 95, 70, and 43 kDa. The in vivo and in vitro results are consistent with the existence of a protein kinase, called NLS kinase, that is specifically activated by NLS peptides.

Purification and N-terminal sequence of a novel interferon-induced 100 kD protein secreted by mouse Ehrlich ascites tumor cells.

Mouse Ehrlich ascites tumor cells secrete a 100 kD protein when they are stimulated by murine interferon-beta (2000 IU/ml). This 100 kD protein was purified from conditioned medium by chromatography on phenyl-Sepharose CL-4B, phosphocellulose, DEAE-Sephacel, and Vydac C4 columns and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single species of 100 kD protein was isolated, and its N-terminal sequence analysis suggested that mouse Ehrlich ascites tumor cell-derived interferon-induced 100 kD protein is a novel protein.

MAPKAP kinase 2 is activated by heat shock and TNF‐α: In vivo phosphorylation of small heat shock protein results from stimulation of the MAP kinase cascade

The activation of MAPKAP kinase 2 was investigated under heat-shock conditions in mouse Ehrlich ascites tumor cells and after treatment of human MO7 cells with tumor necrosis factor-alpha (TNF-alpha). MAPKAP kinase 2 activity was determined using the small heat-shock proteins (sHsps) Hsp25 and Hsp27 as substrates. In both cell types, about a threefold increase in MAPKAP kinase 2 activity could be detected in a time interval of about 10-15 min after stimulation either by heat shock or TNF-alpha. Phosphorylation of MAPKAP kinase 2, but not the level of MAPKAP kinase 2 mRNA, was increased after heat shock in EAT cells. It is further shown that activation of MAPKAP kinase 2 in MO7 cells is accompanied by increased MAP kinase activity. These data strongly suggest that increased phosphorylation of the sHsps after heat shock or TNF-alpha treatment results from phosphorylation by MAPKAP kinase 2, which itself is activated by phosphorylation through MAP kinases. Hence, we demonstrate that MAPKAP kinase 2 is responsible not only for phosphorylation of sHsps in vitro but also in vivo. The findings link sHsp phosphorylation to the MAP kinase cascade, explaining the early phosphorylation of sHsp that is stimulated by a variety of inducers such as mitogens, phorbol esters, thrombin, calcium ionophores, and heat shock.