Mammalian cells that express Bacillus cereus phosphatidylinositol-specific phospholipase C have increased levels of inositol cyclic 1:2-phosphate, inositol 1-phosphate, and inositol 2-phosphate.

Phosphatidylinositol-specific phospholipase C (PtdIns-PLC) of Bacillus cereus catalyzes the conversion of PtdIns to inositol cyclic 1:2-phosphate and diacylglycerol. NIH 3T3, Swiss mouse 3T3, CV-1, and Cos-7 cells were transfected with a cDNA encoding this enzyme, and the metabolic and cellular consequences were investigated. Overexpression of PtdIns-PLC enzyme activity was associated with elevated levels of inositol cyclic 1:2-phosphate (2.5-70-fold), inositol 1-phosphate (2-20-fold), and inositol 2-phosphate (3-20-fold). The increases correlated with the levels of enzyme expression obtained in each cell type. The turnover of phosphatidylinositol (PtdIns) was also increased in transfected CV-1 cells by 13-fold 20 h after transfection. The levels of PtdIns, phosphatidic acid, diacylglycerol, or other inositol phosphates were not detectably altered. Expression of bacterial PtdIns-PLC decreased rapidly after 20 h implying that either the increased PtdIns turnover or the accumulation of inositol phosphates was detrimental to cells and that by some adaptive mechanism enzyme expression was suppressed.     

Expression of transforming growth factor alpha and its messenger ribonucleic acid in human breast cancer: its regulation by estrogen and its possible functional significance

We have studied the estrogenic regulation and the potential autocrine role of transforming growth factor alpha (TGF alpha) in the human breast cancer cell line MCF-7. A biologically active apparent mol wt 30 k TGF alpha was identified by gel filtration chromatography in medium conditioned by MCF-7 breast cancer cells. We previously reported induction of TGF alpha levels in medium by 17 beta-estradiol. We now report correlated increases in TGF alpha mRNA, by Northern and slot blot analysis, after estrogen treatment of MCF-7 cells in vitro. In vivo experiments confirmed these data: estrogen withdrawal from MCF-7 tumor-bearing nude mice resulted in a decline in tumor size and TGF alpha mRNA levels. To explore the functional significance of TGF alpha in MCF-7 cells, anti-TGF alpha antibody was added to MCF-7 soft agar cloning assays. Inhibition of MCF-7 growth resulted, supporting an autocrine role for TGF alpha. Further experiments using an anti-EGF receptor antibody expanded this data, demonstrating inhibition of estrogen-stimulated monolayer MCF-7 cell growth. Examining the generality of TGF alpha expression, 4.8 kilobase TGF alpha mRNAs were seen in three other human breast cancer cell lines, MDA-MB-231, ZR 75B, and T47D. Expression of TGF alpha mRNA was detected in 70% of estrogen receptor positive and negative primary human breast tumors from 40 patients when examined by slot blot and Northern analysis. Thus, we have demonstrated broad expression of TGF alpha in human breast cancer, its hormonal regulation in an estrogen-responsive cell line, and its possible functional significance in MCF-7 cell growth.     

Growth stimulation and differential regulation of transforming growth factor-beta 1 (TGF beta 1), TGF beta 2, and TGF beta 3 messenger RNA levels by norethindrone in MCF-7 human breast cancer cells.

Transforming growth factor-beta (TGF beta) is a potent growth inhibitor in most epithelial cells. We evaluated the effects of norethindrone (which in combination with estrogen is commonly used in oral contraceptives) and other progestins [medioxyprogesterone acetate (MPA) and R5020, which are not used in oral contraceptives] on cell growth and the expression of TGF beta 1, TGF beta 2, and TGF beta 3 mRNAs in MCF-7 human breast cancer cells. Growth of MCF-7 cells was stimulated by norethindrone (10(-8)-10(-5) M), with maximal growth stimulation at 10(-7) M norethindrone after 7 days of treatment. However, the growth of MCF-7 cells was not affected by MPA (10(-8) M) or R5020 (10(-8) M). Treatment with the antiestrogen 4-hydroxytamoxifen at a concentration of 10(-7) M blocked the growth stimulation induced by norethindrone. The norethindrone-induced growth stimulation was accompanied by a dramatic decrease in TGF beta 2 and TGF beta 3 mRNA levels, whereas the level of TGF beta 1 mRNA was not affected by any of the compounds tested. In addition, treatment with MPA or R5020 did not affect TGF beta 2 and TGF beta 3 mRNA levels. The inhibitory effect of norethindrone on TGF beta 2 and TGF beta 3 mRNA levels could be blocked by the addition of 10(-7) M 4-hydroxytamoxifen. Norethindrone as well as estradiol decreased estrogen receptor mRNA levels and increased progesterone receptor mRNA levels. This is the first report which demonstrates that norethindrone stimulates estrogen-responsive human breast cancer cell growth and inhibits the expression of TGF beta 2 and TGF beta 3 mRNAs. These results suggest that the differential regulation of TGF beta expression by norethindrone may be at least partly responsible for the growth stimulation induced by norethindrone. Thus, the norethindrone component of some oral contraceptives may be sufficiently estrogenic to facilitate the development of breast cancer.     

Growth regulatory peptide production by human breast carcinoma cells

The mechanisms by which human breast cancers regulate their own growth have been studied by us in an in vitro model system. We showed that specific growth factors (IGF-I, TGF alpha, PDGF) are secreted by human breast cancer cells. A variety of experiments suggest that they are involved in tumor growth and progression. These activities are induced by estradiol in hormone-dependent breast cancer cells and secreted constitutively by estrogen-independent cells. Concentrates of conditioned medium derived from breast cancer cells can induce the growth of hormone-dependent cells in vivo in athymic nude mice. Hormone-dependent breast cancer cells also secrete TGF beta. TGF beta is growth inhibitory. Growth inhibitors such as antiestrogens or glucocorticoids increase TGF beta secretion. An antiestrogen-resistant mutant of MCF-7 cells does not secrete TGF beta when treated with antiestrogen, but is growth inhibited when treated with exogenous TGF beta. Thus, TGF beta functions as a negative autocrine growth regulator and is probably responsible for some of the growth inhibitory effects of antiestrogens.     

Transforming growth factor-alpha expression is enhanced in human mammary epithelial cells transformed by an activated c-Ha-ras protooncogene but not by the c-neu protooncogene, and overexpression of the transforming growth factor-alpha complementary DNA leads to transformation

MCF-10A cells are a spontaneously immortalized normal human mammary epithelial cell line. MCF-10A cells were transfected with two expression vector plasmids containing either a human point-mutated c-Ha-ras protooncogene or the rat c-neu protooncogene. c-Ha-ras-transfected MCF-10A cells grow as colonies in soft agar, exhibit a 3- to 4-fold increase in their growth rate in serum-free medium, and show a reduced mitogenic response to exogenous epidermal growth factor (EGF) or transforming growth factor-alpha (TGF alpha) as compared to MCF-10A cells. c-Ha-ras-transfected MCF-10A cells express a 4- to 8-fold increase in TGF alpha mRNA levels and secrete 4- to 6-fold more TGF alpha protein as compared to MCF-10A cells. Addition of either an anti-TGF alpha neutralizing monoclonal antibody or an anti-EGF receptor blocking monoclonal antibody to the Ha-ras-transformed MCF-10A cells produces a 50 to 80% inhibition of colony formation of these cells in soft agar. c-neu-transfected MCF-10A cells grown in soft agar and exhibit an increase in their growth rate in serum-free medium at a level comparable to that observed in Ha-ras-transformed MCF-10A cells. Addition of an anti-c-erbB-2 monoclonal antibody inhibits the anchorage-independent growth of these cells in soft agar. However, c-neu-transformed MCF-10A cells show no increase in TGF alpha secretion and no change in their responsiveness to exogenous EGF or TGF alpha. A recombinant retroviral vector containing the human TGF alpha gene was also introduced into MCF-10A cells. TGF alpha-infected MCF-10A cells secrete 15- to 20-fold more TGF alpha protein than MCF-10A cells, form colonies in soft agar, exhibit an enhanced growth rate in serum-free medium, and show a decreased mitogenic response to exogenous EGF or TGF alpha at a level equivalent to Ha-ras-transformed MCF-10A cells. Growth of TGF alpha-infected MCF-10A cells in soft agar is completely inhibited by anti-TGF alpha neutralizing or anti-EGF receptor blocking monoclonal antibodies. These results suggest that TGF alpha is an intermediary in the transformation of human mammary epithelial cells by an activated c-Ha-ras gene, but not by the c-neu gene, and demonstrate that overexpression of this growth factor is able to transform immortalized human mammary epithelial cells which also express a sufficient complement of functional EGF receptors.     

Transforming growth factor beta modulates epidermal growth factor-induced phosphoinositide metabolism and intracellular calcium levels

Transforming growth factor beta (TGF beta) alters the cellular response to epidermal growth factor (EGF) in a number of systems, but the underlying mechanisms for these alterations are largely unknown. We have examined second messenger formation in Rat-1 cells following treatment with EGF and/or TGF beta to determine whether the ability of TGF beta to potentiate some EGF-stimulated processes might be mediated by TGF beta-induced alterations in the signal transduction mechanism. Incubation of serum-deprived confluent Rat-1 cells with 10 ng/ml TGF beta resulted in a marked elevation of cellular inositol trisphosphate and inositol tetrakisphosphate levels, which were maximal at 4 h and maintained for at least 8 h. The effect of TGF beta on levels of inositol trisphosphate and inositol tetrakisphosphate was blocked by actinomycin D, suggesting that RNA synthesis was required for the TGF beta effect. While EGF stimulation induced a rapid and transient (5 min) rise in inositol phosphate levels in control cells, the EGF effect was considerably increased, both in magnitude and duration, by TGF beta treatment. Measurement of intracellular free Ca2+ with fura-2 demonstrated that TGF beta treatment markedly increased the EGF-stimulated rise in free Ca2+ and increased the duration of the response. The positive effects of TGF beta on EGF stimulation could not be explained on the basis of increased EGF binding to cells. We conclude that TGF beta treatment can both activate phosphatidylinositol turnover independently and also sensitize Rat-1 cells to stimulation by EGF.     

Inositol trisphosphate levels in cells expressing wild-type and mutant polyomavirus middle T antigens: evidence for activation of phospholipase C via activation of pp60c-src.

The transforming protein of polyomavirus, middle T (mT), forms a complex with two cellular enzymes: the protein tyrosine kinase pp60c-src and a phosphatidylinositol (PtdIns) 3-kinase. A mutant virus, Py1178T, encodes an mT protein which associates with and activates pp60c-src to the same extent as the wild type but fails to associate with PtdIns 3-kinase. To investigate relationships between activation of pp60c-src, association of PtdIns 3-kinase, and cellular levels of the second messenger inositol 1,4,5-trisphosphate (InsP3), we examined the effects of wild-type and mutant mT proteins on inositol metabolism in rat and mouse fibroblasts. Expression of either wild-type or 1178T mT caused a 300 to 500% increase in the InsP3 level. Cells transformed by Rous sarcoma virus also showed similar increases in InsP3 levels. Mutant mT proteins which failed to activate pp60c-src (NG59 and 1387T) had no effect on InsP3 levels. Pulse-chase experiments with [3H]inositol showed that the turnover of phosphoinositides was increased in cells transformed by either wild-type polyomavirus or Py1178T as compared with the normal parent cell line. The turnover of inositol phosphates was unchanged upon transformation. These data indicate that cells expressing either wild-type or mutant 1178T mT or pp60v-src exhibit elevated levels of InsP3 because of activation of phospholipase C. This activation appears to depend, directly or indirectly, upon activation of pp60src protein kinase activity. Activation of pp60c-src and elevation of InsP3 content are not sufficient for full transformation. Full transformation also requires the association of mT-pp60c-src complexes with PtdIns 3-kinase.     

Review of <Emphasis Type="Italic">Molecular Biology of Human Cancers</Emphasis> (<Emphasis Type="Italic">An Advanced Student’s Text</Emphasis>, by Wolfgang Schulz (Department of Urology and Center for Biological and Medical Research,Heinrich Heine University,Düsseldorf) ISBN 1-4020-3185-8

Postmenopausal women with estrogen receptor positive (ER⁺) breast cancer frequently respond paradoxically to estrogen administration with tumor regression. Using both LTED and E8CASS cells derived from MCF-7 breast cancer cells by long-term estrogen-deprivation, we previously reported that 17 -estradiol (estradiol) is a powerful, pro-apoptotic hormone which kills the cancer cells through activation of the Fas/FasL death receptor pathway. We postulated that the mitochondrial interactive protein Bcl-2 might play a role in the regulation of estradiol-induced apoptosis in both LTED and E8CASS cells. In this study, we assessed estradiol effects on cell growth, proliferation and apoptosis. Additionally we investigated the effect of estradiol on caspase activation, NF-KB and Bcl-2 expression. The functional role of Bcl-2 in estradiol-induced apoptosis was further studied by knockdown or decrease of Bcl-2 with siRNA. Our results show that estradiol significantly inhibited cell growth primarily through a pro-apoptotic action involving caspase-7 and 9 activations (p < 0.01). Basal Bcl-2 and NF-KB levels were greatly elevated and estradiol decreased NF-KB, but not Bcl-2 expression. Knockdown of Bcl-2 expression with siRNA decreased the levels of this protein by 9 fold (p < 0.01). This reduction markedly sensitized both LTED and E8CASS cells to the pro-apoptotic action of estradiol, leading to a synergistic induction of apoptosis and a concomitant reduction in cell number (p < 0.01). Therefore, down-regulation of Bcl-2 synergistically enhanced estradiol-induced apoptosis in ER⁺ postmenopausal breast cancer cells.     

Down-regulation of Bcl-2 enhances estrogen apoptotic action in long-term estradiol-depleted ER<Superscript>+</Superscript> breast cancer cells

Postmenopausal women with estrogen receptor positive (ER⁺) breast cancer frequently respond paradoxically to estrogen administration with tumor regression. Using both LTED and E8CASS cells derived from MCF-7 breast cancer cells by long-term estrogen-deprivation, we previously reported that 17 -estradiol (estradiol) is a powerful, pro-apoptotic hormone which kills the cancer cells through activation of the Fas/FasL death receptor pathway. We postulated that the mitochondrial interactive protein Bcl-2 might play a role in the regulation of estradiol-induced apoptosis in both LTED and E8CASS cells. In this study, we assessed estradiol effects on cell growth, proliferation and apoptosis. Additionally we investigated the effect of estradiol on caspase activation, NF-KB and Bcl-2 expression. The functional role of Bcl-2 in estradiol-induced apoptosis was further studied by knockdown or decrease of Bcl-2 with siRNA. Our results show that estradiol significantly inhibited cell growth primarily through a pro-apoptotic action involving caspase-7 and 9 activations (p < 0.01). Basal Bcl-2 and NF-KB levels were greatly elevated and estradiol decreased NF-KB, but not Bcl-2 expression. Knockdown of Bcl-2 expression with siRNA decreased the levels of this protein by 9 fold (p < 0.01). This reduction markedly sensitized both LTED and E8CASS cells to the pro-apoptotic action of estradiol, leading to a synergistic induction of apoptosis and a concomitant reduction in cell number (p < 0.01). Therefore, down-regulation of Bcl-2 synergistically enhanced estradiol-induced apoptosis in ER⁺ postmenopausal breast cancer cells.     

Elevated expression of the estrogen receptor prevents the down-regulation of p21Waf1/Cip1 in hormone dependent breast cancer cells

Expression of an estrogen receptor alpha (ER) transgene in hormone independent breast cancer and normal breast epithelial cells arrests cell cycling when estradiol is added. Although endogenously expressed ER does not typically affect estradiol-induced cell cycling of hormone dependent breast cancer cells, we observed that elevated expression of a green fluorescent protein fused to ER (GFP-ER) hindered entry of estrogen treated MCF-7 cells into S phase of the cell cycle. In analyses of key cell-cycle regulating proteins, we observed that GFP-ER expression had no affect on the protein levels of cyclin D1, cyclin E, or p27, a cyclin dependent kinase (Cdk) inhibitor. However, at 24 h, p21 (Waf1, Cip1; a Cdk2 inhibitor) protein remained elevated in the high GFP-ER expressing cells but not in non-GFP-ER expressing cells. Elevated expression of p21 inhibited Cdk2 activity, preventing cells from entering S phase. The results show that elevated levels of ER prevented the down-regulation of p21 protein expression, which is required for hormone responsive cells to enter S phase.     

Are polyphosphorylated phospholipids involved in the hormonal control of cholesterol side-chain cleavage activity in tumour Leydig cells?

The possible role of LH or dcAMP induced changes in polyphosphorylated phospholipid metabolism in the regulation of cholesterol side-chain cleavage activity has been studied in tumour Leydig cells. Mitochondria isolated from LH-stimulated Leydig cells were 400% more active in pregnenolone production than mitochondria from control cells. Steroid production in isolated mitochondria from control cells could be stimulated only 25% by cytosol fractions from stimulated cells and 100 microM phosphatidyl inositol-4'-phosphate (PtdIns4P). Other polyphosphorylated phospholipids were either inactive or showed aspecific effects. During a preincubation period tumour cells were labelled with [32P]phosphate and steady-state labelling was obtained for the pholyphosphorylated phospholipids after 40-60 min. [32P]Phosphate incorporation in Ptd Ins4P, phosphatidyl inositol (PtdIns), phosphatidyl choline (PtChl), phosphatidyl ethanolamine (PtdEtn) and cardiolipin (CL) was not affected by treatment of the Leydig cells with LH which stimulated (6-fold), or with cycloheximide which suppressed (4-fold) steroid production. A 25% increase of phosphate incorporation by LH was observed only in phosphatidyl inositol-4',5'-biphosphate (PtdIns4,5P2). 32P Incorporation in PtdIns4,5P2, PtdIns,PtdEtn and CL was stimulated by quinacrine 50 microM. Under these conditions the LH-stimulated pregnenolone production but not the 25-hydroxycholesterol dependent pregnenolone production, was completely inhibited. The results obtained with isolated mitochondria and intact cells indicate that increased levels of polyphosphorylated phospholipids are not consistently correlated with increased mitochondrial pregnenolone production. This argues against an important role of polyphosphorylated phospholipids in the hormonal regulation of cholesterol side-chain cleavage activity in tumour Leydig cells.     

Sex hormone-binding globulin, its membrane receptor, and breast cancer: a new approach to the modulation of estradiol action in neoplastic cells

The role of human Sex Hormone-Binding Globulin (SHBG), the plasma carrier of sex steroids, and its membrane receptor, SHBG-R, in estrogen-dependent breast cancer has been investigated in our laboratory in the past few years. SHBG-R is expressed in MCF-10 A cells (not neoplastic mammary cells), MCF-7 cells (breast cancer, ER positive) and in tissue samples from patients affected with ER positive breast cancer, but not in estrogen-insensitive MDA-MB 231 cells. The SHBG/SHBG-R interaction, followed by the binding of estradiol to the complex protein/receptor, causes a significant increase of the intracellular levels of cAMP, but does not modify the amount of estradiol entering MCF-7 cells. The estradiol-induced proliferation of MCF-7 cells is inhibited by SHBG, through SHBG-R, cAMP and PKA. Similarly, the proliferation rate of tissue samples positive for SHBG-R was significantly lower than the proliferation rate of negative samples. SHBG and SHBG-R could thus trigger a ‘biologic’ anti-estrogenic pathway. In order to get a more detailed knowledge of this system, we first examined the frequence of the reported mutated form of SHBG in 255 breast cancer patients. The mutated SHBG is characterized by a point mutation (Asp 327→Asn) causing an additional N-glycosylation site, which does not affect the binding of steroids to SHBG. The frequence of the mutation was significantly higher (24.5%) in estrogen-dependent breast cancers than in healthy control subjects (11.6%). This observation confirms the close relationship between SHBG and estrogen-dependent breast cancer and suggests that the mutation could modify SHBG activity at cell site. Lastly, the possibility of using SHBG to modulate the estradiol action in breast cancer was further studied by transfecting MCF-7 cells with an expression vector carrying the SHBG cDNA (study in collaboration with G.L. Hammond). Transfected cells are able to produce significant amount of SHBG in their medium, but their SHBG-R is reduced to undetectable levels. The SHBG produced by transfected MCF-7 cells is, however, able to inhibit estradiol-induced proliferation of MCF-7 cells expressing a functional receptor. Thus, the local production of SHBG obtained with transfection could be a useful tool to control cell growth in estrogen-dependent breast cancer.     

Regulation by estrogen through the 5'-flanking region of the transforming growth factor alpha gene.

Expression of transforming growth factor alpha (TGF alpha) mRNA and protein can be stimulated by estrogens such as 17 beta-estradiol (E2) in estrogen-responsive rodent and human breast cancer cells. To ascertain if E2 can directly regulate TGF alpha expression through the 5'-flanking region of the human TGF alpha gene, E2-responsive MCF-7 or ZR-75-1 human breast cancer cells or E2-nonresponsive MDA-MB-231 breast cancer cells were transiently transfected with a plasmid containing an 1140-base pair (bp) Sac-I fragment of the TGF alpha 5'-flanking region ligated to the chloramphenicol acetyltransferase (CAT) gene. Cells that were transfected and subsequently treated with physiological concentrations of E2 (10(-11)-10(-8) M) for 24 h exhibited a 2- to 10-fold increase in CAT activity. The E2 stimulation of CAT activity was dose-dependent with an increase first found at 10(-10) M E2. The increase in CAT activity could be detected within 24-36 h after the addition of E2. There was no significant change in CAT activity in transiently transfected MDA-MB-231 cells as mediated through the TGF alpha 5'-flanking region after E2 treatment. MCF-7 cells were also transiently transfected with different fragments of the TGF alpha 5'-flanking region ligated to the luciferase gene. In the absence of E2 treatment, no detectable luciferase activity was found.(ABSTRACT TRUNCATED AT 250 WORDS)     

Novel functional PI 3-kinase antagonists inhibit cell growth and tumorigenicity in human cancer cell lines.

New efforts in cancer therapy are being focused at various levels of signaling pathways. With phosphoinositide 3-kinase (PI3-K) potentially being necessary for a range of cancer-related functions, we have investigated the influence of selected inositol tris- to hexakisphosphates on cell growth and tumorigenicity. We show that micromolar concentrations of inositol 1,3,4,5,6-pentakisphosphate and inositol 1,4,5,6-tetrakisphosphate [Ins(1,4,5,6)P(4)] inhibit IGF-1-induced [(3)H]-thymidine incorporation in human breast cancer (MCF-7) cells and the ability to grow in liquid medium and form colonies in agarose semisolid medium by small cell lung cancer (SCLC) cells, a human cancer cell line containing a constitutively active PI3-K. In an ovarian cancer cell line that also contains a constitutively active PI3-K (SKOV-3 cells), Ins(1,4,5,6)P(4) again inhibited liquid medium growth. Furthermore, when applied extracellularly, inositol 1,3,4,5-tetrakisphosphate was shown indeed to enter SCLC cells. These effects appeared specifically related to PH domains known to bind to phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P(2)] and phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)], indicating involvement of the PI3-K downstream target protein kinase B (PKB/Akt). This was further supported by inhibition of PKB/Akt PH domain membrane targeting in COS-7 cells by Ins(1,4,5,6)P(4). Thus, we propose that specific inositol polyphosphates inhibit PI3-K by competing with PtdIns(3,4, 5)P(3)-binding PH domains and that this occurs mainly at the level of the downstream PI3-K target, PKB/Akt.     

Inhibition of CDP-DG: inositol transferase by inostamycin.

Inostamycin, a novel microbial secondary metabolite, inhibited [3H]inositol and 32P1 incorporation into phosphatidylinositol (PtdIns) induced by epidermal growth factor (EGF) in cultured A431 cells, the IC50 being 0.5 micrograms/ml, without inhibiting macromolecular synthesis. The drug inhibited cellular inositol phosphate formation only when it was added at the same time as labeled inositol. It was found to inhibit in vitro CDP-DG:inositol transferase activity of the A431 cell membrane, the IC50 being about 0.02 micrograms/ml. It did not inhibit tyrosine kinase, PtdIns phospholipase C, or PtdIns kinase. Therefore, inhibition of PtdIns turnover by inostamycin must be due to the inhibition of CDP-DG:inositol transferase. Thus, inostamycin is a novel inhibitor of CDP-DG:inositol transferase.     

Regulation of extranuclear PtdIns5P production by phosphatidylinositol phosphate 4-kinase 2alpha

Cellular levels of the phosphoinositide PtdIns5P are regulated by agonist stimulation, but the mechanisms controlling turnover of this lipid, and the subcellular location of the regulated PtdIns5P pool(s), remain poorly understood. Here we show that enhanced tyrosine phosphorylation robustly increases cellular PtdIns5P levels. Moreover, unlike PtdIns5P production enhanced by cell stress, we show that this pool of PtdIns5P is specifically regulated by the inositol lipid kinase PIP4K2a.     

Antigen receptor-mediated regulation of sustained polyphosphoinositide turnover in a human T cell line. Evidence for a receptor-regulated pathway for production of phosphatidylinositol 4,5-bisphosphate

Stimulation of the human T cell line, Jurkat, by the addition of monoclonal antibodies reactive with the T cell antigen receptor complex (CD3/Ti) leads to sustained increases in levels of inositol 1,4,5-trisphosphate. To investigate the possibility that the production of polyphosphoinositides is regulated during CD3/Ti stimulation, we studied Jurkat cells whose inositol phospholipids had been labeled to steady state with [3H]inositol, as well as Jurkat cells during nonequilibrium labeling with [32P]orthophosphate. The addition of CD3 monoclonal antibodies led to a 4-5-fold increase in [3H]inositol trisphosphate that was sustained for greater than 20 min. Within 60 s of CD3/Ti stimulation, [3H] phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and [3H]phosphatidylinositol 4-phosphate (PtdIns4P) decreased by 65 and 35%, respectively. This change in [3H]PtdIns(4,5)P2 persisted for greater than 20 min. The decrease in [3H]PtdIns4P, however, was transient, and, after 5 min, the levels of [3H]PtdIns4P were comparable in stimulated and unstimulated cells. To examine the rate of flux through inositol phospholipids, we measured the CD3/Ti-stimulated changes in the ratio, 32P cpm/3H cpm, in each inositol phospholipid. CD3/Ti stimulation led to accelerated fluxes through PtdIns(4,5)P2 and phosphatidylinositol (PtdIns) that were maintained for greater than 20 min. After the initial 30 s, however, there was no detectable effect of anti-CD3 on flux through Ptsins4p. This observation suggested that, during CD3/Ti stimulation, production of PtdIns(4,5)P2 from PtdIns might occur via a small pool of PtdIns4P with a very high turnover. The existence of such a pool was established by determining that, in stimulated cells, the 32P-specific activity of the 1-position phosphate of PtdIns(4,5)P2 was 8-10-fold that of PtdIns4P. We conclude that, during the initial 60 s of CD3/Ti stimulation, there is a substantial depletion of cellular PtdIns(4,5)P2 and PtdIns4P. Thereafter, a CD3/Ti-regulated pathway generates PtdIns(4,5)P2 from PtdIns through a small, but highly labile, pool of PtdIns4P.