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J Blay

Publications and source records attributed to J Blay.

At least 19 recordsLinked to original sources
PubMed2006

Phase 1 European Organisation for Research and Treatment of Cancer study determining safety of pegylated liposomal doxorubicin (Caelyx) in combination with ifosfamide in previously untreated adult patients with advanced or metastatic soft tissue sarcomas.

This phase I study evaluated the toxicity of first-line combined pegylated liposomal doxorubicin (Caelyx) and ifosfamide in patients with advanced and/or metastatic soft tissue sarcomas. Five dose levels (L) were studied: Caelyx 30 mg/m2 (L1-4) or 40 mg/m2 (L5) 1-h infusion d 1 q 3 weeks + ifosfamide and mesna at X g/m2/4 h d 1-3 q 3 weeks at five doses: L1: X = 1.7 g; L2: X = 2 g; L3: X = 2.5 g; L4 and L5: X = 3 g. Cohorts of 3 patients were entered at each level unless a dose-limiting toxicity (DLT) occurred. In case of DLT in 1 of 3 patients a new cohort was added. Toxicity was evaluated by Common Toxicity Criteria (CTC). A total of 28 patients was included: 4 at dose L1, 8 at L2, 3 at L3, 6 at L4, and 7 at L5. Median age was 60 years (range 29-69 years). Male/female ratio was 12/16. Seventy-five percent of patients had a performance status of 1.0 and 36% had leiomyosarcomas. No DLT was observed at dose L1-4. Six patients developed a DLT at dose L5, and thus the recommended dose is level 4 (i.e. Caelyx 30 mg/m2/1 h d 1+ifosfamide at 3 g/m2/4 h d 1-3 q 3 weeks). Few haematological and biochemical events were observed and the principal toxicities were granulocytopaenia and leucopaenia. Five patients discontinued therapy because of toxicity, 4 of them at dose level 5. Non-haematological toxicities > grade 2 were also few. Palmar-plantar erythrodysesthesia (PPE) > grade 1 was not seen. Two patients obtained partial response (PR) and 13 stable disease (SD). Median overall survival (OS) was 333 d and median progression-free survival (PFS) 174 d. In conclusion, this seems to be a feasible combination in patients with advanced soft tissue sarcomas, allowing ifosfamide to be given in a dosage similar to that used when given alone. The recommended dose for future studies is Caelyx 30 mg/m2/1 h d 1+ifosfamide 3 g/m2/4 h d 1-3 q 3 weeks.

Adolescent↗
PubMed2000

Technology evaluation: GEM-231, Hybridon Inc.

Hybridon is conducting studies of the DNA methyltransferase gene and has identified specific sequences on mRNA as targets for chemically-modified antisense oligonucleotides. Hybridon has synthesized compounds that alter methylation of cultured human cancer cells and inhibit their ability to grow in cell culture and inhibit tumor formation in mice [191303]. The work is being carried out in collaboration with McGill University in Montreal and as part of a joint venture called MethylGene, set up by Hybridon and private investors. GEM-231 (a mixed backbone oligonucleotide) is a strand of synthetic DNA, which has been modified with 2'-O-methyl ribose at both ends in order to resemble RNA. It has the ability to inhibit expression of the R1-alpha subunit of protein kinase A, a gene whose expression has been associated with many types of cancer [273331,275860].

Animals↗
PubMed2000

GEM-231 (Hybridon).

GEM-231 is an 18-mer hybrid oligonucleotide under development by Hybridon for the potential treatment of cancer. This compound was initially developed for colon cancer [256660], and progressed to phase II trials in October 1998 [301009]. Hybridon initiated a phase I dose-escalation study enrolling up to 25 patients with refractory solid tumors, in January 1998 at the Lombardi Cancer Center, Georgetown University Medical Center [275860]. GEM-231 was well tolerated in multiple, escalating doses, and that high plasma levels could be safely achieved [301009]. In addition to antitumor effects, when used as a single agent in animal tumor models, GEM-231 has also demonstrated potentiation of the effects of certain conventional cytotoxic chemotherapy drugs [230699]. Hybridon is conducting studies of the DNA methyltransferase gene and has identified specific sequences on mRNA as targets for chemically-modified antisense oligonucleotides. Hybridon has synthesized compounds that alter methylation of cultured human cancer cells and inhibit their ability to grow in cell culture and inhibit tumor formation in mice [191303]. The work is being carried out in collaboration with McGill University in Montreal and as part of a joint venture called MethylGene, set up by Hybridon and private investors. GEM-231 and other oligonucleotides were claimed in WO- 09515378. Hybridon has been issued with two US patents, US- 05652355 and US-05562356, claiming chemically advanced, mixed-backbone oligonucleotides. The first claims mixed backbone 'hybrid' oligonucleotides, which are second generation chemistries, comprising an internal segment of modified DNA flanked by segments of modified RNA (2'-O-methyl substituted). The other claims mixed backbone 'inverted hybrid' oligonucleotides, which comprises an internal segment of naturally-linked, 2'-O-substituted RNA flanked by modified DNA segments [257135].

Journal Article↗
PubMed1999

The adhesion of anti-CD3-activated mouse T cells to syngeneic colon adenocarcinoma cells is differentially regulated by protein tyrosine kinase-, protein kinase C-, and cAMP-dependent pathways in the effector cell.

The adhesion of anti-CD3-activated mouse T cells (AK-T cells) to syngeneic colon adenocarcinoma (MCA-38) cells is mediated principally through the integrin VLA-4 (alpha4beta1). We investigated the signalling pathways through which this adhesive interaction might be regulated. The protein tyrosine kinase inhibitors genistein and methyl 2,5-dihydroxycinnamate (MDHC) markedly inhibited the adhesion of AK-T cells to MCA-38 cells. Furthermore, pretreatment of the AK-T cells alone (but not the MCA-38 targets) with MDHC inhibited adhesion to a comparable extent as when MDHC was present during the assay. Calphostin C, an inhibitor of protein kinase C, also inhibited the adhesion of AK-T cells to MCA-38 monolayers. However, the phosphatidylinositol 3-kinase inhibitor wortmannin failed to alter AK-T cell adhesion to MCA-38 tumour cells. Inhibition of protein kinase A with the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate had no effect on adhesion, but the adenylyl cyclase activator forskolin and the cell-permeable cAMP analogues 8-Br-cAMP and dibutyryl-cAMP significantly suppressed adhesion. Pretreatment of AK-T cells alone with forskolin also inhibited adhesion. The adhesion of AK-T cells to MCA-38 tumour targets is therefore promoted by protein tyrosine kinases and protein kinase C, but inhibited by cAMP-dependent pathways, and the predominant location of the regulatory pathways is within the effector cell.

Adenocarcinoma↗
PubMed1999

Isolation and characterization of an anti-HSV polysaccharide from Prunella vulgaris.

A water soluble substance was isolated from a Chinese herb, Prunella vulgaris, by hot water extraction, ethanol precipitation and gel permeation column chromatography. Chemical tests showed that the substance was an anionic polysaccharide. Using a plaque reduction assay, the polysaccharide at 100 microg/ml was active against the herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), but was inactive against cytomegalovirus, the human influenza virus types A and B, the poliovirus type 1 or the vesicular stomatitis virus. The 50% plaque reduction dose of the polysaccharide for HSV-1 and HSV-2 was 10 microg/ml. Clinical isolates and known acyclovir-resistant (TK-deficient or polymerase-defective) strains of HSV-1 and HSV-2 were similarly inhibited by the polysaccharide. Pre-incubation of HSV-1 with the polysaccharide at 4, 25 or 37 degrees C completely abrogated the infectivity of HSV-1, but pre-treatment of Vero cells with the polysaccharide did not protect cells from infection by the virus. The addition of the polysaccharide at 0, 2, 5.5 and 8 h post-infection of Vero cells with HSV-1 at a multiplicity of infection (MOI) of five reduced the 20 h-yield of intracellular infectious virus by 100, 99, 99 and 94%, respectively. In contrast, a similar addition of heparin showed 85, 63, 53 and 3% reduction of intracellular virus yield, respectively. These results suggest that the polysaccharide may inhibit HSV by competing for cell receptors as well as by some unknown mechanisms after the virus has penetrated the cells. The Prunella polysaccharide was not cytotoxic to mammalian cells up to the highest concentration tested, 0.5 mg/ml and did not show any anti-coagulant activity. In conclusion, the polysaccharide isolated from P. vulgaris has specific activity against HSV and its mode of action appears to be different from other anionic carbohydrates, such as heparin.

Animals↗
PubMed1998

The International Prognostic Index correlates to survival in patients with aggressive lymphoma in relapse: analysis of the PARMA trial. Parma Group.

The objectives of the present study were to investigate the prognostic value of the International Prognostic Index (IPI) at relapse in the 215 patients with intermediate- or high-grade non-Hodgkin's lymphoma (NHL) included in the PARMA trial. The IPI at relapse was available in 204 (95%) of these patients. Response rates to 2 courses of DHAP were 77%, 54%, 55%, and 42% in patients with an IPI of 0, 1, 2 and 3, respectively (P <.02), whereas complete response (CR) rates were 33%, 29%, 20%, and 0% in the same groups of patients (P <.03). With a median follow-up period of 79 months, overall survival (OS) at 5 years was 46%, 25%, 25%, and 11% in these four groups (P <.001). One hundred nine patients responding to 2 courses of DHAP were randomized to receive either BEAC (carmustine, etoposide, cytarabine, cyclophosphamide and mesna) followed by autologous bone marrow transplantation (ABMT) or 4 additional courses of DHAP: IPI at relapse was found highly correlated to OS in patients treated in the DHAP arm (5-year OS: 48%, 21%, 33%, and 0% for IPI 0, 1, 2, and 3, respectively; P =.006), but not in the BEAC arm (5-year OS: 51%, 47%, 50%, and 50% for IPI 0, 1, 2, and 3, respectively; P =.90). OS was significantly superior in the BEAC arm as compared with the DHAP arm in patients with an IPI >0 (P <.05), but not in patients with an IPI of 0. In conclusion, these results show that IPI correlates to response and overall survival in patients with aggressive NHL in relapse and enables us to identify patients with a significantly different outcome among those treated with conventional chemotherapy alone.

Adolescent↗
PubMed1997

Treatment of the P815 murine mastocytoma with cisplatin or etoposide up-regulates cell-surface Fas (CD95) expression and increases sensitivity to anti-Fas antibody-mediated cytotoxicity and to lysis by anti-CD3-activated killer-T cells.

We have investigated the effect of pre-treatment with the anti-cancer drugs cisplatin and etoposide on the susceptibility of P815 murine mastocytoma cells to lysis by murine spleen-derived anti-CD3-activated killer-T (AK-T) cells. A 20 hr pre-treatment with cisplatin (0.2-2 microg/ml) or etoposide (0.01-1 microg/ml) rendered P815 cells significantly more sensitive to AK-T cell-mediated lysis in a 4 hr 51Cr-release assay than untreated control tumor cells. At lower concentrations, pre-treatment with cisplatin or etoposide had no direct cytotoxic effects on P815 tumor cells, as measured by the MTT assay. AK-T cell-mediated killing of P815 tumor cells pre-treated with 2 microg/ml cisplatin or 1 microg/ml etoposide was only partially inhibitable by the Ca2+ chelator EGTA, suggesting that the Ca2+-independent Fas (CD95)/Fas ligand cytolytic pathway of AK-T cells contributes to cytotoxicity. In comparison to untreated control P815 cells, 2 microg/ml cisplatin- or 1 microg/ml etoposide-treated P815 cells exhibited increased expression of Fas mRNA and cell-surface Fas, which correlated with increased sensitivity to lysis by AK-T cells. In addition, pre-treatment with cisplatin or etoposide caused P815 tumor cells to become sensitive to the cytotoxic effects of anti-Fas antibody in a 4 hr 51Cr-release assay. Taken together, our results demonstrate that short-term exposure to concentrations of cisplatin and etoposide in the low cytotoxic range and below up-regulates Fas expression by P815 tumor cells, thereby facilitating cytotoxicity mediated through the Fas/Fas ligand cytolytic pathway.

Animals↗
PubMed1997

The extracellular fluid of solid carcinomas contains immunosuppressive concentrations of adenosine.

The purine nucleoside adenosine (9-beta-D-ribofuranosyladenine) inhibits a number of lymphocyte functions in vitro, including the ability of activated T lymphocytes and natural killer cells to adhere to and kill tumor targets. Solid tumors, such as adenocarcinomas of the lung and colon, are frequently hypoxic and are, therefore, likely to exhibit increased adenine nucleotide breakdown through the 5'-nucleotidase pathway, yielding adenosine. We examined whether the concentration of adenosine in the extracellular fluid of such tumors is adequate to cause immunosuppression. Murine tumors grown in syngeneic hosts or human tumors grown in immunodeficient nu/nu mice were subjected to microdialysis, and adenosine levels in the microdialysate were measured by high-performance liquid chromatography. Treatment of the tumor microdialysates with adenosine deaminase eliminated the adenosine peak. Recovery of adenosine ranged from 15 to 29%, depending on the microdialysis probe, and concentrations of adenosine in tumors ranged from 0.2 to 2.4 microM with a mean of 0.5 microM. In contrast, the adenosine concentration measured s.c. at the same location was 30 +/- 5 nM (mean +/- SE). Inclusion of the adenosine deaminase inhibitor coformycin (10 microM) and the adenosine kinase inhibitor 5'-iodotubercidin (0.1 microM) in the microdialysis perfusion buffer increased extracellular adenosine concentration in tumors to as high as 13 microM. These data show that extracellular adenosine levels in solid tumors are sufficient to suppress the local antitumor immune response and that interference with pathways of adenosine metabolism causes marked increases in tumor extracellular adenosine concentration.

Adenosine↗
PubMed1997

2-chloroadenosine stimulates granule exocytosis from mouse natural killer cells: evidence for signal transduction through a novel extracellular receptor.

The effect of 2-chloroadenosine (2CA), an adenosine receptor agonist, on the activation status of mouse natural killer (NK) cells was determined. Splenic lymphocytes incubated with 2CA exocytosed an NK cell-associated granzyme with N alpha-CBZ-L-lysine thiobenzyl ester (BLT) esterase activity in a dose- and time-dependent manner. Selective depletion of NK cells by anti-asialoGM1 antibody plus complement pretreatment confirmed that NK cells were the source of the BLT esterase activity. 2CA-induced granule exocytosis was not reduced in the presence of the nucleoside uptake blockers NBTI, dilazep, or dipyridamole, indicating the involvement of an extracellular receptor. However, adenosine or other A1, A2, or A3 cell-surface adenosine receptor agonists failed to trigger the exocytotic process. Furthermore, the nonselective adenosine receptor antagonist theophylline, as well as the selective A1 receptor antagonist DPCPX and the selective A2 receptor antagonist DMPX, did not interfere with 2CA-induced BLT esterase secretion. These data suggest that 2CA acts on NK cells via a novel (non-A1/A2/A3) cell-surface receptor. Genistein, a protein tyrosine kinase inhibitor, and calphostin C, a protein kinase C inhibitor, both interfered with 2CA-induced granule exocytosis. Pertussis toxin, an ADP-ribosylating toxin to which certain GTP-binding proteins are sensitive, also inhibited 2CA-stimulated BLT esterase release. In addition, 2CA-induced granule exocytosis was reduced in the presence of cyclosporin A, an inhibitor of Ca(2+)-dependent signaling pathways, and the Ca(2+)-chelating agent EGTA. We conclude that 2CA, acting through a novel extracellular receptor on mouse NK cells, triggers granule exocytosis via a Ca(2+)-dependent signal transduction pathway that is coupled to GTP-binding proteins and involves protein tyrosine kinase and protein kinase C activation.

2-Chloroadenosine↗
PubMed1997

Adenosine acts through a novel extracellular receptor to inhibit granule exocytosis by natural killer cells.

Adenosine concentrations in solid tumors are elevated as a result of altered metabolism by hypoxic cancer cells within the tumor microenvironment. In this study we show that adenosine inhibits mouse natural killer (NK) cell function by interfering with the process of granule exocytosis. Adenosine, at concentrations ranging from 5-25 microM, had a marked inhibitory effect on granule exocytosis by mouse spleen cells stimulated with phorbol myristate acetate and ionomycin. Selective depletion of spleen cell subsets by antibody and complement treatments established that granule exocytosis was mediated by NK cells. Blocking the cellular uptake of adenosine with NBTI or dilazep failed to prevent the inhibitory effect of adenosine, indicating the involvement of a cell-surface receptor. However, adenosine-induced inhibition of granule exocytosis was not blocked by the nonselective A1 and A2 receptor antagonists theophylline and 8-phenyltheophylline, the A1 receptor antagonist DPCPX, or the A2 receptor antagonist DMPX. In addition, the A3 receptor agonists APNEA and NECA failed to affect granule exocytosis. Taken together, these data provide evidence that adenosine inhibits NK cell granule exocytosis by interacting with a novel extracellular receptor. A similar inhibitory effect of tumor-elaborated adenosine on the function of NK cells and other cytotoxic lymphocytes may contribute to tumor survival in the face of host cell-mediated immune defenses.

Adenosine↗
PubMed1995

Use of cultured permanent lines of intestinal epithelial cells for the assay of okadaic acid in mussel homogenates.

Two non-transformed intestinal epithelial cell lines (RIE-1 and IEC-6) and three intestinal carcinoma cell lines (HT-29, T84 and MCA-38) were used to detect the toxic effects due to okadaic acid in the presence of mussel tissue homogenates. Cytotoxicity was assessed by measurement of the metabolic conversion of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide to its corresponding formazan dye. All of the cell lines were sensitive to okadaic acid, with HT-29, RIE-1 and IEC-6 showing the greatest response. Dilutions of whole mussel tissue homogenates caused some loss of viability in most of the cell lines but had no significant effect on the viability of RIE-1 cells. In no case did the mussel homogenate compromise the dose-response curve to okadaic acid when data were normalized to the appropriate control value. These intestinal epithelial cell lines may prove useful in the bioassay of okadaic acid and related toxins.

Animals↗
PubMed1994

Adenosine inhibits the adhesion of anti-CD3-activated killer lymphocytes to adenocarcinoma cells through an A3 receptor.

We have investigated the hypothesis that adenosine, a purine nucleoside produced within hypoxic regions of solid tumors, may interfere with the recognition of tumor cells by cytolytic effector cells of the immune system. We measured the adhesion of murine spleen-derived anti-CD3-activated killer (AK) lymphocytes to syngeneic MCA-38 colon adenocarcinoma cells in a model system. Adenosine, in the presence of the adenosine deaminase inhibitor coformycin to prevent the breakdown of adenosine, inhibited adhesion by up to 60%. The inhibitory effect of adenosine was exerted on the AK cells and not on the MCA-38 targets. The response to adenosine was generated at the cell surface, since the inhibition of adhesion was not abrogated by S-(4-nitrobenzyl)-6-thioinosine or dipyridamole, which block adenosine uptake. The inhibition of adhesion due to adenosine was not blocked by either the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine or the A2 receptor antagonist 3,7-dimethyl-1-propargylxanthine. This suggested that a non-A1, A2 receptor might be involved. The relative order of potencies of adenosine and common analogues was: 5'-N-ethylcarboxamidoadenosine = adenosine = (R)-phenylisopropyladenosine > N6-cyclopentyladenosine > 2-chloro-N6-cyclopentyladenosine = 2-p-(2- carboxyethyl)phenethylamino-5'-N-ethyl-carboxamidoadenosine. This agonist potency profile was again inconsistent with either the A1 or the A2 receptor subtype but indicated that the recently described A3 receptor subtype might be responsible for the inhibition of adhesion. Consistent with this suggestion, aminophenylethyladenosine, an adenosine analogue that binds with high affinity to A3 receptors, inhibited the adhesion of AK cells to MCA-38 tumor cells with high potency (50% inhibitory concentration approximately 1 nM). Adenosine, therefore, interferes with the AK cell recognition of colorectal tumor targets by acting through an A3 receptor on the effector cells. We suggest that this mechanism of immunosuppression, secondary to tissue hypoxia, may be important in the resistance of colorectal and other solid cancers to immunotherapy.

Adenocarcinoma↗
PubMed1994

2-Chloroadenosine inhibits the MHC-unrestricted cytolytic activity of anti-CD3-activated killer cells: evidence for the involvement of a non-A1/A2 cell-surface adenosine receptor.

Adenosine is likely to be a frequent constituent of the tumor microenvironment since this purine nucleoside is produced in quantity by hypoxic cells such as those found in the interior of poorly vascularized solid tumors. In this study we show that 2-chloroadenosine (2CA), a stable analogue of adenosine, inhibits, in a dose-dependent fashion, MHC-unrestricted killing of P815 tumor target cells by anti-CD3-activated killer (AK) lymphocytes. 2CA mediates this effect by interfering with the recognition/adhesion phase of cytolysis. Blocking cellular uptake of 2CA with dipyridamole, rather than attenuating the inhibitory effect, potentiated the inhibition of cytolysis, indicating the involvement of a cell-surface receptor. However, neither the A1 receptor antagonist DPCPX, nor the A2 receptor antagonist DMPX were able to block the inhibitory effect of 2CA on AK lymphocyte function. Similarly, the nonselective A1 and A2 receptor antagonists, theophylline and 8-phenyltheophylline, had no effect on 2CA-mediated inhibition of AK cell activity. Taken together, these data provide evidence that 2CA inhibits the cytolytic activity of AK lymphocytes by interacting with a novel non-A1/A2 cell-surface receptor. A similar effect mediated in vivo by tumor-elaborated adenosine may be involved in tumor-associated immunosuppression.

2-Chloroadenosine↗
PubMed1994

Colon adenocarcinoma cells inhibit anti-CD3-activated killer cell induction.

Adoptive immunotherapy with lymphokine-activated killer (LAK) cells has shown some promise in the treatment of certain cancers that are unresponsive to conventional treatment approaches. However, colon adenocarcinomas tend to respond poorly to LAK therapy, possibly as a result of tumor-induced immunosuppression. Recently, in vivo administration of anti-CD3 antibody has been shown to induce mouse T lymphocytes to mediate major-histocompatibility-complex(MHC)-unrestricted tumoricidal activity which is distinct from natural-killer-cell-derived LAK activity. It has therefore been suggested that anti-CD3 therapy may find application in tumor immunotherapy in humans. However, the effectiveness of anti-CD3-activated killer cell induction within the environment found in the vicinity of colon adenocarcinoma cells has not been evaluated. The present report demonstrates that colon cancer cells of human (HT-29) and mouse (MCA-38) origin markedly inhibit the generation of activated killer cells in murine spleen cell cultures. DNA synthesis and interleukin-2 production by spleen cells following stimulation with anti-CD3 antibody are also profoundly depressed in the presence of MCA-38 and HT-29 adenocarcinoma cells. MCA-38- and HT-29-mediated inhibition of activated killer cell development is exerted through the production of a tumor-associated soluble factor that is distinct from transforming growth factor beta or prostaglandins. Local immunosuppression associated with sites of tumor growth may therefore represent a major obstacle to successful anti-CD3 immunotherapy of certain colon adenocarcinomas.

Adenocarcinoma↗
PubMed1994

Cyclosporin A inhibits 2-chloroadenosine-induced DNA cleavage in mouse thymocytes.

Incubation of mouse thymocytes with the adenosine analogue 2-chloroadenosine resulted in enhanced internucleosomal DNA fragmentation which could be inhibited by the immunosuppressive drug cyclosporin A. In order to be effective, cyclosporin A had to be added to thymocyte preparations at the same time as 2-chloroadenosine. Since cyclosporin A is a selective inhibitor of calcineurin, our data suggest a possible role for calcineurin as a signaling intermediate in the apoptotic pathway activated in thymocytes through adenosine receptors. However, at the present time we cannot exclude the possibility that the inhibitory effect of cyclosporin A on 2-chloroadenosine-induced apoptosis may be mediated through a calcineurin-independent process.

2-Chloroadenosine↗
PubMed1989

Epidermal-growth-factor-stimulated phosphorylation of calpactin II in membrane vesicles shed from cultured A-431 cells.

Membrane vesicles shed from intact A-431 epidermoid carcinoma cells and harvested in the presence of Ca2+ contained epidermal-growth-factor (EGF) receptor/kinase substrates of apparent molecular masses 185, 85, 70, 55, 38 and 27 kDa. The 38 kDa substrate (p38) was recognized by an antibody that had been raised against the human placental EGF receptor/kinase substrate calpactin II (lipocortin I). The A-431 and placental substrates, isolated by immunoprecipitation after phosphorylation in situ, yielded identical phosphopeptide maps upon limited proteolytic digestion with each of five different enzymes. The A-431-cell vesicular p38 is therefore calpactin II. EGF treatment of the intact A-431 cells before inducing vesiculation was not necessary for the substrate to be present within the vesicles. Our data thus indicate that receptor internalization is not a prerequisite for receptor-mediated phosphorylation of calpactin II. The ability of the protein to function as a substrate for the receptor/kinase depended upon the continued presence of Ca2+ during the vesicle-isolation procedure. EGF-stimulated phosphorylation of calpactin II was much less pronounced in vesicles prepared from A-431 cells in the absence of Ca2+, although comparable amounts of the protein were detectable by immunoblotting. Calpactin II therefore appears to be sequestered in a Ca2+-modulated manner within shed vesicles, along with at least four other major targets for the EGF receptor/kinase. The vesicle preparation may be a useful model system in which to study the phosphorylation and function of potentially important membrane-associated substrates for the receptor.

Annexins↗