Inhibitory effect of 8-chloro-cyclic adenosine 3',5'-monophosphate on cell growth of gastric carcinoma cell lines.

A cAMP analogue, 8-chloro-cAMP (8-Cl-cAMP), selectively binds to site 1 receptor of type II regulatory subunit (RII) of cAMP-dependent protein kinase. The effects of 8-Cl-cAMP on human gastric carcinoma cell lines were studied. Twenty microM 8-Cl-cAMP clearly inhibited cell growth in six cell lines (TMK-1, KATO-III, MKN-7, -28, -45, and -74) but not in MKN-1. Cell population in the G1 phase was increased in KATO III cells, which were more responsive to 8-Cl-cAMP, while cell cycle progression in TMK-1 and MKN-1 cells was apparently not influenced by 8-Cl-cAMP. The various changes induced by 8-Cl-cAMP were further analyzed in TMK-1 cells. Decrease of type I regulatory subunit (RI) of cAMP-dependent protein kinase and translocation of RII from cytosol to nucleus were induced by 8-Cl-cAMP treatment. 8-Cl-cAMP increased the level of cAMP-response element (CRE) binding protein in addition to inducing FOS mRNA, whose promoter contains CRE. 8-Cl-cAMP decreased the expression of mRNA for transforming growth factor-alpha (TGF-alpha), while the expression of epidermal growth factor receptor was not changed. Expression of HRAS and MYC mRNAs was slightly increased, whereas the amounts of HRAS and MYC proteins remained unchanged. Our results overall suggest that 8-Cl-cAMP might be a useful tool for antitumor therapy of gastric cancers and that cell growth inhibition by 8-Cl-cAMP might account for the decrease of TGF-alpha expression by tumor cells.     

Epidermal Growth Factor Induces the Expression of Its Receptor Gene in Human Gastric Carcinoma Cell Line TMK-1

To ascertain the possible autocrine pathway in the growth promotion of gastric carcinomas, a study was made on the effects of exogenous human epidermal growth factor (hEGF) on the expression of mRNA for EGF, transforming growth factor-α (TGF-α), EGF receptor, FOS and MYC genes by TMK-1 cells. Exogenous hEGF increased FOS and MYC mRNA levels 30 min and 1 h after the treatment, respectively. TMK-1 cells accumulated the mRNA for EGF receptor about 7- to 8-fold by 3 h after treatment Expressions of mRNA for EGF and TGF-α genes were detected, but the amounts of the mRNA of these genes in TMK-1 cells were not altered after the treatment.     

Alteration of type II regulatory subunit of cAMP-dependent protein kinase in human cisplatin-resistant cells as a basis of collateral sensitivity to 8-chloro-cAMP.

A cyclic adenosine 3',5'-monophosphate (cAMP) analogue, 8-chloro-cAMP (8-Cl-cAMP), had a collateral growth-inhibitory effect on a cis-diamminedichloroplatinum(II) (CDDP)-resistant human cancer cell lines (PC-14/CDDP). The non-selective analogues dibutyryl-cAMP, 8-bromo-cAMP and forskolin, which are cAMP agonists, showed far less cytotoxicity than 8-Cl-cAMP in both cell lines. There was no significant difference in cAMP content between PC-14 and PC-14/CDDP. Because 8-Cl-cAMP has been shown to bind selectively to the site I receptor of the type II regulatory subunit (RII) of cAMP-dependent protein kinase, we determined the level of expression of regulatory subunits in PC-14 and PC-14/CDDP cells by photoaffinity labeling. PC-14/CDDP cells had a higher RII level, low site I receptor of type I regulatory subunit (RI) level, and a lower RI/RII ratio than the parental PC-14 cells. Exposure to 8-Cl-cAMP increased the RI and RII level in PC-14/CDDP cells in dose- and time-dependent manners. On the other hand, in parental PC-14 cells, RII was not detected and the levels of RI and RII were not increased by exposure to 8-Cl-cAMP. These results suggested that the change in RI and/or RII levels caused by 8-Cl-cAMP was correlated with 8-Cl-cAMP-induced growth inhibition and that the collateral sensitivity to 8-Cl-cAMP in CDDP-resistant cells was due to the increased RII level. Our results suggest that 8-Cl-cAMP can be used in combination with CDDP and that measurement of RI and RII levels and/or the RI/RII ratio is a useful tool to predict CDDP sensitivity.     

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.     

Expression of Interleukin-6 and Its Effect on the Cell Growth of Gastric Carcinoma Cell Lines

The expression and the effect of IL-6 were examined in human gastric carcinoma cell lines to determine whether IL-6 serves as a growth stimulator. The expression of IL-6 mRNA was detected in three (TMK-1, MKN-1, MKN-7) of 8 gastric carcinoma cell lines. All three cell lines secreted IL-6 into the culture fluid, in large amounts in the cases of MKN-1 and MKN-7 cells. Scatchard plot analysis of IL-6 binding revealed that MKN-1 and MKN-7 cells had both high- and low-affinity receptors. Cell growth of MKN-1 and MKN-7 cells was stimulated by IL-6, while anti-IL-6 antibody inhibited growth. The expression of IL-la mRNA by these three cell lines was induced by IL-6. IL-la increased the expression of mRNA for IL-6 by TMK-1 cells. These findings indicate that IL-6 induced by IL-la is an autocrine growth factor for some gastric carcinomas     

Induction of Growth Factor-receptor and Metalloproteinase Genes by Epidermal Growth Factor and/or Transforming Growth Factor-α in Human Gastric Carcinoma Cell Line MKN-28

We examined the effects of epidermal growth factor (EGF) and transforming growth factor-α (TGF-α) on EGF receptor (EGFR) phosphorylation and the expression of mRNAs for oncogenes, growth factors, their receptors and metalloproteinase genes by MKN-28 gastric carcinoma cells which express EGF, TGF-α and EGFR genes. Both EGF and TGF-α stimulated EGFR phosphorylation. EGF and TGF-α induced FOS, MYC and ERBB-2 oncogene expression. Interestingly, EGF increased the expression of mRNAs for TGF-α and EGFR. On the other hand, TGF-α increased TGF-α mRNA but decreased the expression of mRNAs for EGFR and TGF-β. Furthermore, mRNAs for interstitial collagenase, stromelysin and procollagen type I genes were also enhanced after treatment with EGF and TGF-α. These results indicate that EGF and TGF-α successively evoke cascade phenomena which favor tumor progression, invasion and extracellular matrix formation, acting as autocrine growth regulators for gastric carcinomas.     

Alteration of Type II Regulatory Subunit of cAMP-dependent Protein Kinase in Human Cisplatin-resistant Cells as a Basis of Collateral Sensitivity to 8-Chloro-cAMP

A cyclic adenosine 3',5'-monophosphate (cAMP) analogue, 8-chloro-cAMP (8-Cl-cAMP), had a collateral growth-inhibitory effect on a cis -diamminedichloroplatinum(II) (CDDP)-resistant human cancer cell lines (PC-14/CDDP). The non-selective analogues dibutyryl-cAMP, 8-bromo-cAMP and forskolin, which are cAMP agonists, showed far less cytotoxicity than 8-Cl-cAMP in both cell lines. There was no significant difference in cAMP content between PC-14 and PC-14/CDDP. Because 8-Cl-cAMP has been shown to bind selectively to the site I receptor of the type II regulatory subunit (RII) of cAMP-dependent protein kinase, we determined the level of expression of regulatory subunits in PC-14 and PC-14/CDDP cells by photoaffinity labeling. PC-14/CDDP cells had a higher RII level, low site I receptor of type I regulatory subunit (RI) level, and a lower RI/RII ratio than the parental PC-14 cells. Exposure to 8-Cl-cAMP increased the RI and RII level in PC-14/CDDP cells in dose- and time-dependent manners. On the other hand, in parental PC-14 cells, RII was not detected and the levels of RI and RII were not increased by exposure to 8-Cl-cAMP. These results suggested that the change in RI and/or RII levels caused by 8-Cl-cAMP was correlated with 8-Cl-cAMP-induced growth inhibition and that the collateral sensitivity to 8-Cl-cAMP in CDDP-resistant cells was due to the increased RII level. Our results suggest that 8-Cl-cAMP can be used in combination with CDDP and that measurement of RI and RII levels and/or the RI/RII ratio is a useful tool to predict CDDP sensitivity.     

Reduced Levels of Transforming Growth Factor-beta Type I Receptor in Human Gastric Carcinomas

The expressions of transforming growth factor beta (TGF-β) and its receptor and TGF-β inhibitory element (TIE)-binding protein were examined on human gastric carcinomas by Northern blot hybridization, immunohistochemistry, affinity labeling and gel retardation analysis. TGF-β mRNA was expressed in tumor and normal tissues at various levels. Immunohistochemically, TGF-β expression was confirmed to be present within tumor cells. Out of the 17 human gastric carcinoma tissues, 14 (82%) showed a reduction in the level of type I receptor (65 kDa) for TGF-β when compared to corresponding normal mucosas. Interestingly, in seven of the 14 tumors the level of TIE-binding protein in the tumor tissue was lower than that in normal mucosa. Human gastric carcinoma cell line TMK-1, whose growth was inhibited by TGF-β, had only type I receptor for TGF-β and showed a high level of TIE-binding protein. Conversely, MKN-1, a TGF-β -resistant cell line, exhibited an extremely low level of TGF-β receptor and had no TIE-binding protein. These results overall indicate that although human gastric carcinoma cells produced TGF-β, they showed a reduction in TGF-β type I receptor and a low level of TIE-binding protein, resulting in escape from growth inhibition by TGF-β.     

Inhibition of growth and modulation of gene expression in human lung carcinoma in athymic mice by site-selective 8-Cl-cyclic adenosine monophosphate

Site-selective cyclic AMP (cAMP) analogues inhibit growth and induce changes in morphology in a spectrum of human cancer cell lines (D. Katsaros et al., FEBS Lett., 223:97, 1987). The cellular events underlying such effects of cAMP analogues include differential regulation of type I versus type II cAMP-dependent protein kinase isozymes (S. Ally et al., Proc. Natl. Acad. Sci. USA, 85: 6319, 1988). Infusion (i.p.) of 8-Cl-cAMP, the most potent site-selective cAMP analogue, for 7 days produced regression of LX-1 lung carcinoma in athymic mice in a dose-dependent manner. The tumor regression correlated with the changing levels of cAMP receptor proteins, RI alpha and RII beta, the regulatory subunits of cAMP-dependent protein kinase type I and type II, respectively. By photoaffinity labeling with 8-N3-[32P]cAMP and immunoblotting with a monospecific anti-RII antibody, RI alpha (Mr 49,000) and RII beta (Mr 51,000) were identified in the untreated control tumors. 8-Cl-cAMP treatment induced a rapid increase of both RI alpha and RII beta in tumor cytosols and translocation (within 1 h) of only RII beta from the cytosol to the nucleus. RII beta in both cytosols and nuclei remained elevated during 8-Cl-cAMP treatment, whereas RI alpha in the cytosols gradually decreased with time of treatment after its initial transient increase. Northern blot analyses demonstrated that the RII beta mRNA level increased within 6 h of 8-Cl-cAMP treatment and remained elevated during treatment, whereas the RI alpha mRNA level decreased to below that of the untreated control tumor level after its transient increase during 1-6 h of treatment. 8-Cl-cAMP treatment also caused a sharp decrease in both N-ras and c-myc mRNA levels. These results suggest that the fundamental basis for the antineoplastic activity of 8-Cl-cAMP may reside in the restoration of normal gene regulation in neoplasms in which cAMP receptor proteins play a role.     

Expression of Amphiregulin, a Novel Gene of the Epidermal Growth Factor Family, in Human Gastric Carcinomas

The expression of mRNA for amphiregulin (AR), a novel gene of the epidermal growth factor family, was examined in 8 human gastric carcinoma cell lines and 32 gastric carcinoma tissues as well as corresponding normal mucosa. Of the 8 gastric carcinoma cell lines, 7 expressed 1.4 kb AR mRNA at various levels. The expression of AR mRNA by TMK-1 and MKN-28 cells was increased by treatment with epidermal growth factor or transforming growth factor α. In surgical cases, all the gastric carcinoma tissues and their adjacent normal mucosa expressed AR mRNA. Interestingly, 20 (62.5%) out of 32 tumors expressed AR mRNA at higher levels than their corresponding normal mucosas (tumor/normal ≥1.2). No obvious correlation was observed between the AR mRNA levels and the histological types or tumor staging of gastric carcinoma. Immunohistochemically, AR protein was localized to the cytoplasm and/or nucleus in tumor cells. These results suggest that AR produced by tumor cells may be involved in the pathogenesis and/or progression of human gastric carcinoma.     

8-chloroadenosine 3',5'-monophosphate as a novel modulator of multidrug-resistance

Multidrug resistance (MDR) is a major obstacle in the chemotherapy of cancer. 8-chloroadenosine 3',5'-monophosphate (8-Cl-cAMP), a site-selective analog of cAMP, produced a potent growth inhibition in a spectrum of MDR cell lines. The IC50 (concentration inhibiting 50% of cell proliferation) of 8-Cl-cAMP at 6 days ranged from 0.1 to 3.0 muM in both P-glycoprotein (pgp)-associated and pgp-unassociated MDR cells, and the growth inhibition occurred with continued cell viability. Growth inhibition paralleled down-regulation of RIalpha subunit and catalytic activity of cAMP-dependent protein kinase. 8-Cl-cAMP also provoked the suppression of the promoter activity of the MDR1 gene. These results demonstrate that type I cAMP-dependent protein kinase plays a role in drug resistance and that 8-Cl-cAMP is a novel modulator of multidrug resistance.     

Differentiation therapy of cancer targeting the ri-alpha regulatory subunit of cAMP-dependent protein-kinase (review)

Consideration of the cancer process as a problem of blocked ontogeny makes attractive the approach to the control of cancer through differentiation therapy. The use of antisense strategy and retroviral vector-mediated gene transfer technology provided direct evidence that two isoforms of cAMP receptor protein, the RI and RII regulatory subunits of cAMP-dependent protein kinase, have opposite, roles in cell growth and differentiation, RI being growth stimulatory while RII is a growth-inhibitory and differentiation-inducing protein. As RIalpha expression is enhanced during cell transformation, and in primary human tumors and cancer cell lines as compared to their normal counterparts, it is an attractive target for cancer treatment. 8-Cl-cAMP and RIalpha antisense oligodeoxynucleotide, those that effectively down-regulate RIalpha and upregulate RIIbeta without producing cytotoxicity, provide new approaches toward differentiation therapy of cancer.     

Unhydrolyzable analogues of adenosine 3':5'-monophosphate demonstrating growth inhibition and differentiation in human cancer cells.

A set of adenosine 3':5'-monophosphate (cAMP) analogues that combine exocyclic sulfur substitutions in the equatorial (Rp) or the axial (Sp) position of the cyclophosphate ring with modifications in the adenine base of cAMP were tested for their effect on the growth of HL-60 human promyelocytic leukemia cells and LS-174T human colon carcinoma cells. Both diasteromeres of the phosphorothioate derivatives were growth inhibitory, exhibiting a concentration inhibiting 50% of cell proliferation of 3-100 microM. Among the analogues tested, Rp-8-Cl-cAMPS and Sp-8-Br-cAMPS were the two most potent. Rp-8-Cl-cAMPS was 5- to 10-fold less potent than 8-Cl-cAMP while Sp-8-Br-cAMPS was approximately 6-fold more potent than 8-Br-cAMP. The growth inhibition was not due to a block in a specific phase of the cell cycle or due to cytotoxicity. Rp-8-Cl-cAMPS enhanced its growth-inhibitory effect when added together with 8-Cl-cAMP and increased differentiation in combination with N6-benzyl-cAMP. The binding kinetics data showed that these Sp and Rp modifications brought about a greater decrease in affinity for Site B than for Site A of RI (the regulatory subunit of type I cAMP-dependent protein kinase) and a substantial decrease of affinity for Site A of RII (the regulatory subunit of type II protein kinase) but only a small decrease in affinity for Site B of RII, indicating the importance of the Site B binding of RII in the growth-inhibitory effect. These results show that the phosphorothioate analogues of cAMP are useful tools to investigate the mechanism of action of cAMP in growth control and differentiation and may have practical implication in the suppression of malignancy.     

Adenovirus-mediated transfer of wild-type p53 gene results in apoptosis or growth arrest in human cultured gastric carcinoma cells.

We examined the susceptibility of six human gastric carcinoma cell lines to infection with recombinant p53 adenovirus vector (AxCA-p53). AxCA-p53 infection at a muliplicity of infection (MOI) of 50 resulted in apoptotic cell death (MKN-1 cells), growth arrest (MKN-45, MKN-74 and KATO-III cells), or non-effectiveness (TMK-1 and OCUM-2M cells). Western blot analysis revealed increasing expression levels of p21/WAF1 protein after infection with AxCA-p53 in all the cell lines. After infection with AxCA-p53, the expression levels of bax or bcl-XL protein changed in MKN-1, but not in the other cell lines. These results suggest that the apoptotic pathway (dependence on the expressions of bcl-2 family proteins) dominates the growth arrest pathway (dependence on the expressions of p21/WAF1 protein) after infection with AxCA-p53. Thus, the bcl-2 family might play a crucial role in p53-mediated growth arrest and apoptosis in human gastric carcinoma cells.     

Synergistic inhibition of growth of breast and colon human cancer cell lines by site-selective cyclic AMP analogues

Our past studies on the mechanism of cyclic AMP (cAMP)-mediated control of tumor growth, using the experimental rat mammary tumor models as well as human breast cancer cell lines, indicated that the action of cAMP is mediated by the RII cAMP receptor protein, the regulatory subunit of cAMP-dependent protein kinase type II (Y. S. Cho-Chung, J. Cyclic Nucleotide Res., 6: 163, 1980). We now shown that the site-selective cAMP analogues, which are manyfold more active in binding to the cAMP receptor protein than previously studied analogues, demonstrate a potent growth inhibition of seven breast and three colon human cancer cell lines. The cAMP receptor protein has two different cAMP binding sites, and cAMP analogues that selectively bind to either one of the two binding sites are known as either site 1 selective (C-8 analogues) or site 2 selective (C-6 analogues). Nineteen site-selective analogues, C-6 and C-8 monosubstituted and C-6,-8 disubstituted, were tested for their growth regulatory effect. The majority of these analogues demonstrated an appreciable growth inhibition, with no sign of toxicity in all 10 cancer lines at micromolar concentrations. The three most potent inhibitors were 8-Cl-, N6-benzyl-, and N6-phenyl-8-thio-p-chlorophenyl-cAMP, demonstrating 50% growth inhibition at 5-25 microM concentrations (IC50). Furthermore, N6-analogues, in combination with halogen or thio derivatives of C-8 analogues, demonstrated synergistic enhancement of growth inhibition. The growth inhibition paralleled a change in cell morphology, an augmentation of the RII cAMP receptor protein, and a reduction in p21 ras protein. The growth inhibition by 8-Cl-cAMP was not due to its metabolite, 8-Cl-adenosine, since: (a) the growth inhibition by 8-Cl-cAMP was released upon cessation of treatment, whereas that by 8-Cl-adenosine was not released; (b) 8-Cl-cAMP treatment did not affect cell cycle progression, whereas 8-Cl-adenosine brought about G1 synchronization; (c) 8-Cl-cAMP treatment caused reduction of p21 ras protein, whereas 8-Cl-adenosine did not affect p21 levels; and (d) 8-Cl-adenosine was not detected in either cell extracts or medium from the cells treated with 8-Cl-cAMP for 48-72 h. Site-selective cAMP analogues thus provide a new physiological means to control the growth of breast and colon human cancer cells.     

Down-regulation of cobalt-induced HIF-1alpha expression correlates with cell proliferation and apoptosis in human gastric carcinoma cells

Previously we reported that the hypoxia-inducible factor-1alpha (HIF-1alpha) expression correlated with cell proliferation and apoptosis under 500 mM of CoCl2 treatment in a human gastric carcinoma cell line, MKN-1. Herein we report a similar correlation in other cell lines, MKN-45 and TMK-1. The dual-phase expression of HIF-1alpha was highest at 6 and 8 h of treatment in MKN-45 and TMK-1, respectively, while the peak in MKN-1 occurred at 4 h. The cell viability indices showed a similar dual phase to the HIF-1alpha expression, while the apoptotic indices started to increase as the level of the HIF-1alpha expression decreased. In our previous study, the FACS analysis showed a marked G2/M arrest and an increase of the pre-G1 area in MKN-1 after 36 h of treatment, whereas the G2/M arrest was not observed in MKN-45 and TMK-1. The expression of cell cycle and apoptosis-related proteins showed a correlation with the HIF-1alpha expression and the FACS results, which suggested that the level of HIF-1alpha correlated with proliferation and apoptosis in human gastric carcinoma cell lines with a possible cell-type specific pattern.     

Epidermal growth factor induces the expression of its receptor gene in human gastric carcinoma cell line TMK-1

To ascertain the possible autocrine pathway in the growth promotion of gastric carcinomas, a study was made on the effects of exogenous human epidermal growth factor (hEGF) on the expression of mRNA for EGF, transforming growth factor-a (TGF-a), EGF receptor, FOS and MYC genes by TMK-1 cells. Exogenous hEGF increased FOS and MYC mRNA levels 30 min and 1 h after the treatment, respectively. TMK-1 cells accumulated the mRNA for EGF receptor about 7- to 8-fold by 3 h after treatment. Expressions of mRNA for EGF and TGF-a genes were detected, but the amounts of the mRNA of these genes in TMK-1 cells were not altered after the treatment.     

Genetic Status and Expression of the Cyclin-dependent Kinase Inhibitors in Human Gastric Carcinoma Cell Lines

Deregulation of cyclin, cyclin-dependent kinases (CDKs) and their inhibitors could have a pivotal role in the development of diverse human cancers. We examined the genetic status and the expression of CDK inhibitors ( p21, p27, pl6 and p15 ), CDK2 and cyclins (A, D1 and E) in eight gastric carcinoma cell lines, in comparison with the status of p53 gene alterations. All the cell lines (except MKN-28) that contained a p53 gene abnormality expressed very low or undetectable levels of p21 mRNA, while the cell lines (MKN-45 and -74) with wild-type p53 gene expressed high levels of p21 mRNA. An inverse correlation was found between the level of p21 mRNA and the expression of mRNAs for CDK2 and G1 cyclins. MKN-28 was an exception; it contained mutated p53 , and expressed mRNAs for p21 , CDK2 and G1 cyclins at high levels. Only MKN-45 and -74, with wild-type p53 , expressed considerable levels of p21 protein. Homozygous deletion of the p16 and p15 genes was detected in two (MKN-45 and HSC-39) of the eight gastric carcinoma cell lines. p16 protein was not expressed in three cell lines (MKN-28, MKN-74 and KATO-III), as well as MKN-45 and HSC-39. Rearrangement of the p15 gene was found in TMK-1. Rearrangement of the p27 gene was detected in MKN-45, although the expression of p27 protein was well preserved in all the gastric carcinoma cell lines. The expression of pRb was also preserved in all the cell lines except KATO-III. No obvious correlation was observed between the p53 gene status and the expression of p27 and p16 . These findings suggest that abnormal regulation of CDK2/cyclins and CDK inhibitors might be involved in deregulated growth of gastric carcinomas.