Decreased expression of type II protein kinase C in HL-60 variant cells resistant to induction of cell differentiation by phorbol diester

To evaluate the molecular basis for susceptibility of the cell differentiation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), we examined biochemical activities and expression of subspecies of protein kinase C from HL-60 cells that are susceptible to differentiation induced by TPA and HL-60R cells, HL-60 variant cells that are resistant to such induction. Analysis of the subcellular distribution of protein kinase C revealed that the activity of this kinase in the cytosol from HL-60R cells was 30% of that from parental HL-60 cells but that the enzyme activities in the membrane showed similar values in these cells. Treatment of HL-60 cells with 100 nM TPA for 30 min resulted in a 75% decrease in protein kinase C activity in the cytosol and a 300% increase in this activity in the membrane. A minor subcellular redistribution of the enzyme activity was found in HL-60R cells after TPA treatment. Based on analysis of protein kinase C isozymes by hydroxyapatite column chromatography, the relative activities of types I, II, and III in the cytosol of HL-60 cells were 11, 80, and 9%, whereas those in HL-60R cells were 27, 36, and 37%, respectively. Type II isozyme was a major protein kinase C in the cytosol of HL-60 cells, but type II was less in the HL-60R cells. Among the three isozymes, type II enzyme was most sensitive to TPA with histone H1 as the substrate, although all three isozymes were activated Ca2+-dependently in the presence of phosphatidylserine. We suggest that the acquired resistance of HL-60R cells toward induction of cell differentiation by TPA may be associated with a decrease in the expression of the type II isozyme of protein kinase C.     

Characterization of the cyclic adenosine 3':5'-monophosphate effector system in hormone-dependent and hormone-independent rat mammary carcinomas

We have compared the properties of cyclic adenosine 3':5'-monophosphate (cAMP)-dependent protein kinases I and II in hormone-dependent/cAMP-sensitive (DMBA tumor) and hormone-independent/cAMP-resistant (DMBA 1 tumor) rat mammary carcinomas. cAMP-resistance was not due to less total kinase in the hormone-independent tumor, grossly altered distribution between soluble and particulate forms of the kinase (80% soluble in either tumor), alteration in the relative proportion of isozymes I and II of the protein kinase (the soluble and the particulate fraction from both tumors contained about 50% of either isozyme), or a decreased sensitivity towards cAMP (both isozymes had affinities for cAMP and its derivatives that corresponded closely with those of isozymes from normal tissues). Furthermore, the sensitivity of the enzymes towards thermal denaturation was identical for samples from the two tumor types. Subtle differences did, however, exist between the regulatory moieties [regulatory subunit of cAMP-dependent protein kinase II (RII)] of isozyme II from the two tumors: autophosphorylated RII from the hormone-independent tumor migrated as a doublet corresponding to Mrs 54,000 and 52,000 on sodium dodecyl sulfate-polyacrylamide gels, against Mrs 53,000 and 52,000 for RII from the hormone-dependent tumor; RII from the two tumors showed different elution profiles upon DEAE-cellulose chromatography; a considerable proportion of the soluble RII in the hormone-independent tumor formed supramolecular aggregates as judged by size-exclusion chromatography. No such microheterogeneity was noted for isozyme I. This study thus shows that the lack of cAMP-responsiveness of one tumor is related either to a defect distal to the cAMP-dependent protein kinases or to the appearance of the new subtype of RII in the resistant tumor. If the latter explanation is correct, it means that the part of the RII molecule responsible for interaction with other proteins rather than that responsible for cAMP-binding and control of protein kinase activity modulates the growth-inhibiting response to cAMP.     

Protein kinase C isozyme expression in human leukemia-lymphoma cell lines--an immunocytochemical study.

There have been an increasing number of reports describing a pivotal role for phosphorylation in cellular responses for cell differentiation and proliferation. We examined an immunocytochemical expression of protein kinase C(PKC) isozymes (type I, II, and III) in 22 leukemia-lymphoma cell lines. Of these cell lines, 21 expressed type II PKC and 17 showed the co-expression of both types II and III PKC in varying degree. The cell line without PKC activity showed far less [3H]-TdR uptake and no heterotransplantation in nude mice. Types II and III PKC appear to relate to cell proliferation in certain leukemia-lymphoma cell lines.     

Level of cyclic AMP-dependent protein kinase isozyme in normal liver and hepatoma tissue and the effect of sodium selenite

Sodium selenite in normal saline was administered intraperitoneally (1 mg/kg) into mice bearing ascitic hepatocarcinoma for 4 days. The cyclic AMP-dependent protein kinase isozymes (type I and type II) in normal liver and hepatocarcinoma cells were separated and assayed. The results show that the level of type I/II is markedly higher in hepatocarcinoma than in the normal liver cells. Sodium selenite is able to reduce it towards the normal level. Further analysis shows that the chief function of sodium selenite is to reduce the raised level of type I/II in hepatocarcinoma cells which, in fact, is due to the increase of total amount of type I cyclic AMP-dependent protein kinase. This paper presents the speculation that one of the mechanisms of the inhibitory effect of sodium selenite on carcinogenesis may be due to the selective action of this compound on the cyclic AMP-dependent protein kinase isozymes in tumor cells, thus inhibiting cancer cell division and facilitating differentiation and reversion.     

Distinct isozyme patterns of cyclic nucleotide phosphodiesterase in human neuroblastoma and ganglioneuroma; a possible marker of differentiation of neural crest-derived tumors and Schwann cells

By DEAE-cellulose chromatography, the 30,000g supernatants of human neuroblastoma (n = 7), ganglioneuroma (n = 5), sympathetic ganglia (n = 3), and Schwannoma (n = 2) were found to contain three major peaks of adenosine 3',5'-cyclic monophosphate (cAMP) phosphodiesterase (PDE) activity, which were termed peaks I, II, and III in the order of their elution from the column. Peak I isozyme was calmodulin-dependent, and had two different Km values for cAMP (32 and 2.3 microM) and a low Km for guanosine 3',5'-cyclic monophosphate (cGMP) (2.9 microM). Peak II isozyme had a high Km for both cAMP, 76 microM, and cGMP, 32 microM, and peak III isozyme was a cAMP-PDE with Km of 1.8 microM. The peak II and III isozymes were calmodulin-independent. The activity ratio of peak I isozyme to peak III isozyme (I/III isozyme ratio) was significantly different (P less than 0.001) in neuroblastoma and in ganglioneuroma/sympathetic ganglia, i.e., 0.23 +/- 0.11 for neuroblastoma vs. 0.79 +/- 0.20 for ganglioneuroma and 0.51 +/- 0.08 for sympathetic ganglia. Schwannoma showed the highest value of 1.05 (P less than 0.05). These results suggest that the I/III isozyme ratio of cAMP-PDE could be a useful marker in studies on the differentiation of neural crest-derived tumors and Schwann cells.     

Cyclic adenosine 3':5'-monophosphate-dependent protein phosphorylation and the control of leukemia L1210 cell growth

Synergistic increases in the survival of mice bearing an L1210 leukemia tumor have been demonstrated previously after treatment with 1,3-bis(2-chloroethyl)-1-nitrosourea together with theophylline over those treated with either agent alone. These results imply that manipulation of cyclic adenosine 3':5'-monophosphate (cyclic AMP) levels in L1210 cells may result in alteration of sensitivity to chemotherapy and alterations in tumor growth. In the present study, we have shown that in vivo treatment of L1210 cells with theophylline results in changes in intracellular cyclic AMP-dependent protein kinase activity levels as well as in an apparent redistribution of both the nuclear and cytoplasmic isozymes. Biochemical events in the tumor cells immediately after administration of theophylline in vivo or a cyclic AMP analog (8-parachlorophenylthio cyclic adenosine 3':5'-monophosphate in vitro were independent of the presence of 1,3-bis(2-chloroethyl)-1-nitrosourea. The changes apparently involve signal transduction via the adenylate cyclase system and manifest as: (a) increased sensitivity of cyclic AMP-dependent protein kinase to activation by cyclic AMP after treatment of L1210 cells with theophylline; (b) decrease in endogenous nuclear protein phosphorylation sites; and (c) protein kinase isozyme redistribution between nuclear and extranuclear compartments, i.e., a relative increase of the type I isozyme activity in the nuclear and of the type II isozyme activity in the 900 x g supernatant fractions after treatment of the mice with theophylline. The relative activity increases are accompanied by a relative decrease of type II activity from the nucleus and type I isozyme activity from the 900 x g extranuclear supernatant fraction. These events appear temporally related to changes in nuclear RNA metabolism as evidenced by altered kinetics of RNA precursor uptake and incorporation into tumor cell RNA after treatment. These results imply that the cyclic AMP-dependent phosphorylative modification of intracellular proteins may play a regulatory role in tumor cell growth and in theophylline-mediated tumor regression.     

Distribution and properties of type I and type II binding proteins in the cyclic adenosine 3':5'-monophosphate-dependent protein kinase system in Wilms' tumor

We compared the relative amounts and properties of cyclic adenosine 3':5'-monophosphate (cAMP)-binding proteins in surgical specimens of Wilms' tumor and normal kidney. Cytosolic fractions of both tissues contained type I and type II isozymes of cAMP-dependent protein kinase (adenosine triphosphate: protein phosphotransferase, EC 2.7.1.37). Among tumor samples, the mean ratio of type I to type II cAMP-binding activity was 2.76 +/- 0.52 (S.D.) contrasted with 1.36 +/- 0.23 for normal kidney (p less than 0.001). The total soluble cAMP-binding activities in normal and malignant tissues differed only slightly. Photoaffinity labeling of cytosol from either tissue, using cyclic adenosine 3':5'-[8-azido-32P]monophosphate, disclosed three cAMP-binding proteins (Mr 47,000, 51,000, and 55,000) that were identified as regulatory subunits of the holoenzyme. Three lower-molecular-weight proteins with unknown function were considered to be proteolytic products of the larger proteins. The Mr 47,000 protein, a monomeric regulatory subunit of type I kinase, was clearly the dominant protein in tumor specimens, but it was much less abundant in normal kidney. The temperature sensitivities of the cAMP-binding proteins and their dissociation constants for cyclic adenosine 3':5'-[8-azido-32P]monophosphate incorporation did not differ appreciably between tumor and normal tissues. Wilms' tumor appears to have a full complement of regulatory subunits of cAMP-dependent protein kinase that are capable of normal cellular function.     

Determination and characterization of hexokinase in thyroid cancer and benign neoplasms

Hexokinase (ADP: D-hexose-6-phosphotransferase, EC 2.7.1.1) was studied in human thyroid carcinomas (n = 11), follicular adenomas (n = 32), and normal thyroid tissue (n = 21). The specific activity was significantly increased in carcinoma (0.163 +/- 0.083 U/mg protein) compared with normal tissue (0.030 +/- 0.010 U/mg protein) (P less than 0.001). Specific activities of follicular adenomas are rather heterogeneous, but when subdivided into three groups according to histopathologic criteria, a significant difference was found between follicular adenomas group I and II and follicular adenomas group III. A lesser cellular differentiation of adenomas is indicated by the lower degree or even absence of colloid production and follicle formation. A higher proliferation rate may be assumed on the grounds of the irregularities in outline, the often defective pseudocapsule, and signs of compression of the surrounding tissue. The highest specific activity in adenomas was found in the group with the highest proliferative activity, i.e., group III, whereas the lowest specific activities were found in adenomas with the lowest grade of proliferation, i.e., group I; the former was comparable with values found in carcinomas and the latter was comparable with values found in normal thyroid tissue. An interesting difference was found when the compartmentation of hexokinase was compared in carcinomas of different degree of differentiation. In papillary carcinomas a significantly lower proportion of hexokinase (HK) is present in the cytosol in comparison to follicular and undifferentiated carcinomas. In carcinomas more HK II and less HK I was found in comparison with normal thyroid tissue. In contrast hexokinase isozyme composition and compartmentation in adenomas were not different from normal thyroid tissue.     

Multiple forms of protein kinase from normal human brain and glioblastoma

The biochemical characteristics of the protein kinase (PK; adenosine triphosphate-protein phosphotransferase, EC 2.7.1.37) isozymes in subcellular preparations from normal human brain cortex and glioblastoma were investigated after chromatography on diethylaminoethyl cellulose, and the following results have been obtained. Two major isozyme forms, eluted by 50 and 200 mM phosphate buffer, are present in both cytosol and membrane-derived preparations from cerebral cortex. Furthermore, these isozyme forms have properties similar to those referred to as type I and type II cyclic adenosine 3':5'-monophosphate-dependent PK. In these chromatographic isozymes, cyclic adenosine 3';5'-monophosphate is more active in stimulating the basal PK enzyme than is cyclic guanosine 3':5'-monophosphate. In glioblastoma, the PK activity from cytosol and particulate preparations is resolved by diethylaminoethyl cellulose in four peaks. In cytosol, the major portion of the enzyme is eluted with a 300 mM buffer (about 50% of the total basal PK activity) and is cyclic nucleotide dependent. On the contrary, in glioblastoma particulate, the PK enzyme is mainly eluted at 50 and 100 mM buffer; neither of these isozymes is cyclic nucleotide dependent. As for cytosol, only the particulate isozyme eluted at 300 mM buffer is strongly activated by cyclic nucleotides. Finally, in both glioblastoma subcellular preparations, only a type II cyclic adenosine 3':5'-monophosphate-dependent PK is present.     

Thermal effects on DNA synthesis in bone marrow cells of patients with acute myeloblastic leukemia.

It has been known that thymidylate synthetase (TS) and thymidine kinase (TK) are DNA-synthesizing enzymes via the de novo and salvage pathways, respectively, in pyrimidine metabolism, and an immunological method using a monoclonal antibody to bromodeoxyuridine (BrdU) is useful for the detection of S-phase cells. We investigated the effects of hyperthermia on DNA synthesis in bone marrow cells obtained from patients with acute myeloblastic leukemia. TK isozymes in bone marrow cells of patients with acute myeloblastic leukemia were separated into two types, i.e., fetal type isozyme in cytosolic cell fraction and adult type isozyme in mitochondrial cell fraction. Heating at over 43 degrees C as a hyperthermia markedly suppressed enzyme activity of fetal type TK isozyme and number of S-phase cells labelled with BrdU, but not activities of adult type TK isozyme and TS. These results indicate that hyperthermia may regulate the proliferation of S-phase cells by the suppression of salvage synthesis of DNA.     

Location of adult and fetal aldolases A, B, and C by immunoperoxidase technique in LF fast-growing rat hepatomas.

The resurgence of aldolase isozymes in cancerous tissues is a well-known but poorly understood phenomenon. This resurgence poses the problem of whether or not adult and fetal aldolase isozymes are produced by the same cells. For clarification of this question, the immunoperoxidase technique was used to locate aldolases A, B, and C in one type of fast-growing hepatoma, the LF hepatoma and, by comparison, in normal adult liver. Under optical microscopy, aldolases A and C were located in the cytoplasm of almost all of the cancerous cells. An isozyme antigenically identical with aldolase B was also demonstrated to be present in almost all of the cells, but the reaction indicating the presence of this isozyme was weaker. In normal adult liver, only aldolases A and B were demonstrated to be present in almost all the hepatocytes. Under electron microscopy in LF hepatoma, the three isozymes were found to be present mainly in the cytoplasm. These facts suggest that the three types of aldolase are very probably present in the same cells at the same time, and they provide indirect arguments leading us to think that the resurgence of fetal aldolase isozymes in cancer is not the consequence of cellular selection but is due to a disturbance at the gene control level.     

Expression of three major protein kinase C isozymes in various types of human leukemic cells

We examined the levels of protein kinase C (PKC) activity and the expressions of its three major isozymes, designated types I (gamma), II (beta), and III (alpha), in the cytosol and particulate fractions of cells from patients with acute myelogenous leukemia (AML), acute lymphocytic leukemia (ALL), and chronic lymphocytic leukemia (CLL), in an attempt to elucidate the cell type- or lineage-specific expression of these isozymes. The levels of PKC activities in the cytosol and particulate fractions from AML cells were higher than those from ALL or CLL cells. The average PKC activities of AML cells, ALL cells, and CLL cells were 18.7, 12.2, and 11.3 pmol/min/10(8) cells, respectively, in the cytosol fractions and 4.4, 3.1, and 2.6 pmol/min/10(8) cells, respectively, in their particulate fractions. M1 cells (French-American-British classification) and AML cells with T-lymphocyte-associated surface antigens, such as CD2 and CD7, had significantly lower PKC activities among AML cells. Immunoblot analyses using monoclonal antibodies against each isozyme revealed that all three isozymes were broadly distributed on leukemic cells with considerable variability in the level of expression. All lymphoid leukemic cells expressed PKC-gamma in the cytosol fractions, albeit a minor component; however, this type was observed in cells from only half the number of AML patients. Those AML cells with cytosolic PKC-gamma usually expressed lymphoid surface antigens, such as CD2, CD7, and CD19. On the other hand, cytosolic PKC-beta and PKC-alpha were commonly observed in all types of leukemic cells. AML cells expressed these two types at almost equal levels, but in lymphoid cells, expressions of PKC-beta were usually more abundant than those of PKC-alpha. These data suggest that AML cells with lymphoid antigens might have a lower PKC activity but more predominant expression of cytosolic PKC-gamma than the usual AML cells.     

Characterization of cyclic adenosine 3':5'-monophosphate-dependent protein kinase isozymes in normal and neoplastic fetal rat brain cells

Fetal brain cells from rats given a transplacental pulse of N-ethyl-N-nitrosourea progressively acquire malignant characteristics and dedifferentiate when grown in vitro. One aspect of this dedifferentiation is a decreased morphological response to cyclic adenosine 3':5'-monophosphate (cAMP). In the present study, we have characterized and compared the isozymes (I, II) of cAMP-dependent protein kinase in fetal brain cells and in the neoplastically transformed, dedifferentiated BT5C glioma cell line. This is a first approach to find the mechanism behind the subresponsiveness of such cells towards cAMP. It is also part of a broader investigation of the cAMP effector system in cells showing various rates of normal and malignant growth. We found the regulatory and catalytic subunits of cAMP-dependent protein kinase to be expressed to a similar degree in both cell types. Sixty % of the enzyme was located in the 30,000 X g supernatant. The glioma cell line had a significantly higher ratio (1.2) between protein kinase I and II than did the normal fetal cells (0.5). This difference in isozyme distribution was not apparent using conventional methods for enzyme separation and detection, the use of specific antibodies being essential for that purpose. Of the chromatographically separated forms (a, b) of protein kinase II, Form IIa was selectively decreased in the glioma cell line. The alterations of the protein kinases in the glioma cell line described above may be of importance for some of the neoplastic properties of these cells. However, the subdued response of such cells towards cAMP is not explained since the concentrations of cAMP or its analogues required for activation of the kinases were similar for the enzymes from normal and neoplastically transformed cells.     

Phosphofructokinase in normal thyroid tissue and thyroid neoplasms

Phosphofructokinase (PFK; ATP: D-fructose-6-phosphate transferase, EC 2.7.1.11) was studied in human thyroid carcinomas (n = 16), follicular adenomas (n = 31) and normal thyroid tissue (n = 19). The specific activity in carcinomas (0.129 +/- 0.070) is significantly increased (p less than 0.001) in comparison with phosphofructokinase in normal thyroid tissue (0.028 +/- 0.009). No difference in phosphofructokinase activity seems to exist between follicular, papillary and undifferentiated carcinomas. Specific activities of follicular adenomas are rather heterogeneous. When these tumors were divided into three groups of increasing proliferative activity as judged by histopathological criteria, highest specific activities of phosphofructokinase were found in the group with the highest proliferative activity. The latter group resembles the enzyme activities found in carcinomas. On the other hand specific enzyme activities of the least active tissues were comparable to normal and different from carcinomas. Adenomas show the same isozyme composition as found in normal thyroid tissue. All three isozymes of PFK, M-(muscle)type, L-(liver)type and P-(platelet)type, were present. Phosphofructokinase from papillary carcinomas show a lesser degree of precipitation with anti-M antibodies. A higher amount of platelet type isoenzyme is found in papillary carcinomas compared to follicular and undifferentiated carcinomas. The influence of citrate, an inhibitor of phosphofructokinase, is in agreement with the isozyme composition.     

Does axitinib (AG-01376) have a future role in metastatic renal cell carcinoma and other malignancies?

Axitinib (Pfizer Inc., UK) is an oral small-molecule receptor tyrosine kinase inhibitor that targets angiogenesis. Axitinib has greater affinity and is a more selective inhibitor of VEGF receptor 1, -2 and -3, PDGFR and c-KIT than both sunitinib and sorafenib. It has encouraging efficacy and safety data in Phase II trials for metastatic renal cell carcinoma and advanced thyroid cancer patients. It is now being investigated in two Phase III trials in metastatic renal cell carcinoma and in Phase II trials in a range of tumor types. These trials will determine whether axitinib is an effective and safe antiangiogenic therapy.     

Targeting the protein kinase C family: are we there yet?

Protein kinase C (PKC) comprises a family of serine/threonine kinases that are involved in the transduction of signals for cell proliferation, differentiation, apoptosis and angiogenesis. Unsurprisingly, disruption of PKC regulation is implicated in tumorigenesis and drug resistance. PKC function is complex in this context owing to the differing roles of individual isozymes within the cell and across tumour types. Therapeutically targeting PKC isozymes is not new; however, with many of the early PKC inhibitor cytotoxic drug combinations being discarded at the phase II level, and recent phase III studies in non-small-cell lung cancer proving negative, what's going wrong?     

Pyruvate kinase isozymes and dedifferentiation of (neuro-)ectodermal tumors

This paper describes the isozyme composition and regulatory properties of pyruvate kinase (PK) from well-differentiated (DMTC) and undifferentiated (AMTC) medullary thyroid carcinomas of the rat. These tumors were chosen as an animal model for human (neuro-)entodermal neoplasms differing in their degree of differentiation. The results were compared with human medullary thyroid carcinomas (MTC) and phaeochromocytomas. AMTC were characterized by increased PK activity, a higher apparent S0.5 for phosphoenolpyruvate, enhanced inhibition by alanine, presence of predominantly M2-type isozyme and loss of M1-type-containing hybrids as compared to DMTC. The alterations in PK isozyme expression and hence kinetic behaviour could not be demonstrated in human MTC or phaeochromocytomas due to the apparently well-differentiated nature of these tumors and the presence of M2-type isozymes. These results are discussed with reference to the nature and significance of PK isozyme shifts found in other tumors. It is suggested that the determination of PK isozyme composition might prove useful in the diagnosis of nueroectodermal neoplasms originating from tissues not primarily expressing M2-type isozyme(s).     

Expression of two types of receptor for insulin-like growth factors in human malignant glioma

Two types of receptors for insulin-like growth factors (IGFs) were characterized in glioma cell lines established from different human brain tumors of glial origin (astrocytoma grades III and IV) by competitive binding assay, affinity labeling, and protein phosphorylation. Type I IGF receptor is a heterodimer composed of alpha-subunits (Mr 130,000), which bind IGF I and II with equal affinity, and of beta-subunits (Mr 98,000), which show tyrosine kinase activity and autophosphorylation stimulated by IGF I and II with equal potency. The type II IGF binding site is a monomer (Mr 250,000) which binds IGF II with 10 times higher affinity than IGF I. The cellular concentration of type II IGF binding site is about 2- to 5-fold higher than the amount of type I IGF receptor. The characteristics of the two types of IGF receptors in human glioma cell lines are similar to those described recently in fetal rat astrocytes. In contrast the type I IGF receptor in glioma cells is different from that studied previously in normal adult brain regarding the equal affinity for IGF I and II, and the higher molecular size of the alpha-subunit (130,000 versus 115,000). It is suggested that glioma cells may represent a fetal cell type in tumor development of adult human brain. A role of IGF in malignant glioma has not yet been determined, but the presence of IGF receptors is a prerequisite for cellular actions of IGF.     

Exploitation of protein kinase C: a useful target for cancer therapy

Protein kinase C is a family of serine/threonine kinases. The PKC family is made up of at least 12 isozymes, which have a role in cell proliferation, differentiation, angiogenesis, and apoptosis. Activation of PKC isozyme is dependent on tyrosine-kinase receptors and G-protein-coupled receptors. PKC isozymes regulate multiple signaling pathways including PI3-K/Akt, MAPK, and GSK-3beta. PKC isozymes have variable roles in tumor biology which in part depend on the cell type and intracellular localization. PKC isozymes are commonly dysregulated in the cancer of the prostate, breast, colon, pancreatic, liver, and kidney. Currently, several classes of PKC inhibitors are being evaluated in clinical trials and several challenges in targeting PKC isozymes have been recently identified. In conclusion, PKC remains a promising target for cancer prevention and therapy.     

Growth inhibition of transforming growth factor beta on human gastric carcinoma cells: receptor and postreceptor signaling.

The effect of transforming growth factor beta (TGF-beta) on human gastric carcinoma cell lines was examined. Cell growth and DNA synthesis of TMK-1 were inhibited by TGF-beta, whereas MKN-28 presented no response to TGF-beta. Scatchard plot analysis of TGF-beta binding showed that TMK-1 had a relatively small number of high-affinity receptors, whereas MKN-28 had a large number of low-affinity receptors. By affinity labeling, only the type I receptor (Mr 65,000) for TGF-beta was detected in TMK-1, while three types of receptors, type I, type II (Mr 85,000-95,000), and type III (Mr 250,000-350,000), for TGF-beta were present in MKN-28. TGF-beta treatment reduced p34cdc-2 kinase activity and the level of phosphorylation of retinoblastoma protein in TMK-1, whereas it did not affect them in MKN-28. mRNAs for MYC and platelet-derived growth factor B chain were increased by treatment of TGF-beta on TMK-1. cAMP-responsive element binding activity was decreased by TGF-beta treatment in MKN-28 but not in TMK-1. This was closely correlated with protein kinase C activity. These results suggest that the type I receptor for TGF-beta in human gastric carcinoma cells may be mainly linked with the growth inhibition of TGF-beta by a decrease in retinoblastoma protein phosphorylation by p34cdc-2 without suppression of MYC expression. Conversely, TGF-beta may reduce protein kinase C activity and cAMP-responsive element binding activity in TGF-beta-resistant gastric carcinoma cells.