Paramedic rapid sequence intubation in patients with non-traumatic coma.

INTRODUCTION: Pre-hospital intubation by paramedics is widely used in comatose patients prior to transportation to hospital, but the optimal technique for intubation is uncertain. One approach is paramedic rapid sequence intubation (RSI), which may improve outcomes in adult patients with traumatic brain injury. However, many patients present to emergency medical services with coma of non-traumatic cause and the role of paramedic RSI in these patients remains uncertain. METHODS: The electronic Victorian Ambulance Clinical Information System was searched for the term 'suxamethonium' between 2008 and 2011. We reviewed the patient care records and included patients with suspected non-traumatic coma who were treated and transported by road-based paramedics. Demographics, intubation conditions, vital signs (before and after drug administration) and complications were recorded. Younger patients (<60 years) were compared with older patients. RESULTS: There were 1152 paramedic RSI attempts of which 551 were for non-traumatic coma. The success rate for intubation was 97.5%. There was a significant drop in blood pressure in younger patients (<60 years) with the mean systolic blood pressure decreasing by 16 mm Hg (95% CI 11 to 21). In older patients, the systolic blood pressure also decreased significantly by 20 mm Hg (95% CI 17 to 24). Four patients suffered brief cardiac arrest during pre-hospital care, all of whom were successfully resuscitated and transported to hospital. CONCLUSIONS: Paramedic RSI in patients with non-traumatic coma has a high procedural success rate. Further studies are required to determine whether this procedure improves outcomes.

Cyclic AMP receptor triggers nuclear protein phosphorylation in a hormone-dependent mammary tumor cell-free system.

Adenosine 3',5'-monophosphate (cyclic AMP) receptor protein of 56,000 daltons increases markedly in mammary tumors induced by 7,12-dimethylbenz[a]anthracene (DMBA) after incubation of tumor slices with cyclic AMP, benzamide, and arginine. Incubation of cytosol from these tumor slices with nuclei from unincubated tumors results in nuclear uptake of the 56,000-dalton cyclic AMP receptor and in phosphorylation of the 76,000-dalton nuclear protein. Binding of the 56,000-dalton receptor and phosphorylation of the 76,000-dalton protein also occur in DMBA tumor nuclei when protein kinase type II of bovine heart is used. The results suggest that cyclic AMP receptor is involved in the nuclear events of a hormone-dependent mammary tumor.

Growth arrest and morphological change of human breast cancer cells by dibutyryl cyclic AMP and L-arginine.

The growth in vitro of human breast cancer cells, line MCF-7, was inhibited by a daily supplement of L-arginine (1 milligram per milliliter). Arginine acted synergistically with dibutyryl adenosine 3',5'-monophosphate (cyclic AMP) (10(-6) molar) to enhance the growth inhibitory effect: the cell replication ceased completely within 2 days after treatment. The growth arrest accompanied a change in cell morphology and was preceded by increases in the cellular concentration of cyclic AMP, adenylate cyclase, and type II cyclic AMP-dependent protein kinase activities as well as a decrease of estrogen binding activity. The results suggest that growth of human breast cancer cells is subject to cyclic AMP-mediated regulation and that arginine may play a specific role in this process.

Relationship of hormone dependency to estrogen receptor and adenosine 3',5'-cyclic monophosphate-binding proteins in rat mammary tumors.

Estrogen-binding activity and the binding activity of adenosine 3',5'-cyclic monophosphate (cAMP) were assayed in the cytosol of biopsy specimens from 70 rat mammary tumors. The response of these tumors to host ovariectomy was followed. The hormone dependency of the tumors was closely related to the relative concentrations of estrogen receptor (ER) and cAMP-binding protein (CR) in the tumor cytosol. When ER and CR were expressed as the ER/CR ratio, 36 of 38 (95%) hormone-dependent tumors had ratios of 35 X 10(-3) or more, whereas 31 of 32 (97%) hormone-independent tumors had ratios of less than 35 X 10(-3). When ER alone was measured, hormone dependency could be correctly predicted in only 60% of the tumors because the ER range for individual tumors of both types greatly overlapped. Ratios of ER/CR were also elevated in normal estrogen target tissues as compared to those in normal estrogen nontarget tissues. Thus determination of the ER/CR ratio in tumor cytosols appeared to be a more reliable method of predicting hormone dependency than was determination of ER alone.

Cyclic adenosine 3',5'-monophosphate-binding protein: role in ovariectomy-induced regression of a hormone-dependent mammary tumor in Sprague-Dawley female rats.

Cyclic AMP- and estrogen-binding activities were present in 7,12-dimethylbenz[a]anthracene-induced mammary carcinomas in Sprague-Dawley female rats. Soon after ovariectomy of the host, cyclic AMP binding markedly increased and estrogen binding decreased in regressing tumors. These changes were reversed when tumor growth was resumed following the injection of estradiol-17beta. The data suggested the possible involvement of cyclic AMP binding in the growth control of a hormone-dependent mammary tumor.

Prognostic significance of the expression of a ras protein with a molecular weight of 21,000 by human breast cancer.

Expression of the cellular ras Mr 21,000 protein (p21) has been measured in tumors from breast cancer patients who at time of presentation had no evidence of metastatic disease. Western blotting analysis revealed that 37 of 54 (69%) tumors contained p21 levels 2- to 10-fold greater than those of control breast tissues. An excessive increase of p21 (5- to 10-fold over the control value) occurred more frequently in tumors of T3 and T4 stages [15 of 25 (60%)] than in tumors at T2 stage [6 of 29 (21%)], suggesting a correlation between advancement of disease and high p21 levels. p21 levels were positively related to the involvement of axillary lymph nodes at the time of primary treatment. As no correlations were detected between p21 levels and a gross pathological parameter, tumor grade, it is possible that p21 levels may reflect the degree of cellular malignancy. This is supported by data on tumor recurrence; 13 of 16 patients (81%) with tumors expressing low p21 levels were disease free for greater than or equal to 4 years after primary treatment, whereas only 5 of 9 patients (56%) with high p21 tumors remained disease free. These results suggested that a quantitative enhancement of p21 oncogene protein is associated with both the progression and prognosis of breast cancer.

Inverse relation between estrogen receptors and cyclic adenosine 3':5'-monophosphate-binding proteins in hormone-dependent mammary tumor regression due to dibutyryl cyclic adenosine 3':5'-monophosphate treatment or ovariectomy.

During the growth arrest of 7,12-dimethylbenz(alpha) anthracene-induced rat mammary carcinomas following ovariectomy or N6, O2'-dibutyryl cyclic adenosine 3':5'-monophosphate (DBcAMP) treatment, a change in the specific estrogen and cAMP binding to tumor proteins is observed. Three days after ovariectomy or DBcAMP treatment of the hosts, cAMP binding increases 5- and 2-fold in the nuclei and cytosol of tumors, respectively, whereas nuclear and cytoplasmic estrogen binding decreases by 70 and 25%, respectively. These changes in cAMP- and estrogen-binding activities are detectable within 1 day after ovariectomy or DBcAMP treatment, and the changes are reversed when resumption of tumor growth is induced by the injection of estradiol valerate or cessation of DBcAMP treatment. When 7,12-dimethylbenz(alpha)anthracene-induced tumors fail to regress after ovariectomy or DBcAMP treatment, the change in estrogen and cAMP binding does not occur. Concomitant with the increase of cAMP-binding activity in regressing tumors are increases in histone kinase activity and the cAMP content of the tumors. These increases in cAMP-binding and protein kinase activities are blocked by cycloheximide. These data suggest an interaction between a steroid hormone and cAMP in the growth control of a hormone-dependent mammary tumor.

Anticarcinogenic effect of N6,O2'-dibutyryl cyclic adenosine 3':5'-monophosphate on 7,12-dimethylbenz(a)anthracene mammary tumor induction in the rat and its relationship to cyclic adenosine 3':5'-monophosphate metabolism and protein kinase.

A single intubation of 7,12-dimethylbenz(a)anthracene (DMBA) (20 mg in 1 ml sesame oil) to female Sprague-Dawley rats at 50 days of age produces primary mammary carcinomas in 80% of rats at 100 to 150 days of age. Administration of N6,O2'-dibutyryl cyclic adenosine 3':5'-monophosphate (DBcAMP) p.o. beginning at 1 day prior to DMBA intubation resulted in marked delay and reduction of tumor production: only 15% as many DBcAMP-treated rats had tumors as in the control group (DMBA only) with 60 days of delay in the first tumor appearance. DMBA-induced tumor production was preceded by changes in the cyclic adenosine 3':5'-monophosphate (cAMP) metabolism and protein kinase of the mammary gland. Within 24 hr post-DMBA intubation, the intracellular cAMP level and adenylate cyclase activity increased with an increase in type I isozyme of cAMP-dependent protein kinase, a form which has been associated with increased proliferative activity and a less differentiated cellular state in other tissues. The increases in cAMP level, adenylate cyclase activity, and the protein kinase activity were transient, and the values decreased to below the control values by Day 10 post-DMBA intubation. In mammary glands of rats that had received DBcAMP, the cAMP level and protein kinase isozyme pattern were similar to those of older rats that are no longer susceptible to the carcinogen. The inhibitory effect on DMBA-induced carcinogenesis may be related to the modifications that DBcAMP induces on cAMP level, adenylate cyclase activity, and cAMP-dependent protein kinase of the mammary gland.

Arrest of hormone-dependent mammary cancer growth in vivo and in vitro by cholera toxin.

Growth of 7,12-dimethylbenz(alpha)anthracene-induced mammary carcinoma in rat was arrested by daily s.c. injections of cholera toxin. At a dose of 2 micrograms/200-g rat, tumors regressed to 50% of their initial size within 2 weeks, and 85% of tumors regressed completely within 4 to 5 weeks. The same response to cholera toxin was observed with another hormone-dependent mammary tumor, MTW9, but not with the hormone-independent tumors, DMBA No. 1 and MT 13762. The latter result was consistent with the lack of response of these hormone-independent tumors to hormone removal (ovariectomy) or N6,O2'-dibutyryl cyclic adenosine 3':5'-monophosphate treatment. The growth-inhibitory effect of cholera toxin was dose dependent, and upon cessation of treatment tumors resumed growth; after complete regression, however, tumors did not reappear until 6 months after termination of the treatment. An amount of cholera toxin as high as 10 micrograms/day/200-g rat s.c. injected over a 6-week period showed no systemic toxicity in the animals. The growth of human breast cancer cells (MCF-7) in culture was also inhibited by a daily supplement of cholera toxin. At a concentration of 100 ng/ml, the cell replication ceased completely within 2 days. The growth inhibitions, both in vivo and in vitro, were accompanied by marked increases in the cellular cyclic adenosine 3':5'-monophosphate content and type II cyclic adenosine 3':5'-monophosphate-dependent protein kinase activity as well as a decrease of estrogen binding activity. Thus, extinction of mammary cancer can be achieved by cholera toxin, an agent that stimulates the intracellular cyclic adenosine 3':5'-monophosphate system.

Cyclic adenosine 3':5-monophosphate receptor proteins in hormone-dependent and -independent rat mammary tumors.

The molecular species of cyclic adenosine 3':5'-monophosphate (cAMP) receptor proteins (high-affinity-binding proteins) present in hormone-dependent and -independent rat mammary carcinomas were identified and characterized. Three major types of cAMP receptor proteins, with molecular weights of 39,000, 48,000, and 56,000, specifically incorporated the photoaffinity label, 8-azido-cyclic adenosine 3':5'-[32P]monophosphate and were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the tumor cytosols. The M.W. 48,000 and 56,000 receptor proteins appeared to be the regulatory subunits of cAMP-dependent protein kinase types I and II, respectively, and the M.W. 39,000 receptor protein was the proteolytic fragment of the M.W. 56,000 receptor protein. The relative amounts of these cAMP receptor proteins varied from one tumor type to another and showed no correlation with respect to the hormone dependency of tumors. Under two-dimensional gel electrophoresis, however, the M.W. 56,000 receptor protein from hormone-dependent tumors migrated as a doublet and shifted to either a more acidic or more basic charge than that of the receptor protein of hormone-dependent tumors. The alteration of the charge of the receptor did not affect the affinity for cAMP binding, because both hormone-dependent and hormone-independent tumor cytosols exhibited the dissociation constant for cAMP of approximately 10(-8) M. The M.W. 56,000 cAMP receptor protein from hormone-dependent tumors exhibited self-phosphorylation, but that from hormone-independent tumors did not. The diethylaminoethyl cellulose elution profiles of cAMP receptor proteins also differed between hormone-dependent and -independent tumors; cAMP binding activity from hormone-dependent tumors coeluted with cAMP-dependent protein kinase activity, whereas most of the cAMP binding activity from hormone-independent tumors eluted at a higher ionic strength than did cAMP-dependent protein kinase activity. These results suggest that the charge alteration of cAMP receptor proteins, which appears to occur at a site remote from that of cAMP binding, may be associated with the hormone independency of mammary tumors.

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.

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.

Autotaxin (NPP-2), a metastasis-enhancing motogen, is an angiogenic factor.

Autotaxin [ATX (NPP-2)], originally isolated as a tumor motility-stimulating protein, has recently been shown to augment tumor aggressiveness. Specifically, atx-transfected, ras-transformed NIH3T3 cell lines have been shown to be more invasive, tumorigenic, and metastatic than mock-transfected ras-transformed control cells. In addition, the atx-transfected ras-transformed cell lines appeared to produce tumors that were much more hyperemic than those formed by appropriate control cells. This observation led to the present study, in which we demonstrate that ATX modulates angiogenesis both directly and indirectly. We have used a murine in vivo angiogenesis model in which treated Matrigel plugs are injected s.c. into athymic nude BALB/c mice. Using the same transfected cell lines as before, we found that mixing atx-transfected ras-transformed NIH3T3 cells into the Matrigel resulted in greater new blood vessel formation than control cells. Similarly, mixing purified ATX into the Matrigel resulted in new blood vessel formation within the plug, similar to that produced by vascular endothelial growth factor. Mechanistically, ATX is not a strong chemoattractant for human endothelial cells (HUVECs); however, it strongly stimulates motility in human coronary artery smooth muscle cells. In addition, ATX stimulates HUVECs grown on Matrigel to form tubules, much like vascular endothelial growth factor. Both of these normal cell types are shown to express and secrete ATX. In HUVECs, ATX expression is up-regulated by basic fibroblast growth factor in a time-dependent manner. This up-regulation also extends to secretion of enzymatically active protein, as demonstrated by Western blot analysis and quantification of type-1 phosphodiesterase activity. These results establish the presence of ATX in HUVECs and coronary artery smooth muscle cells and specify ATX as a novel angiogenic factor, suggesting that ATX could contribute to the metastatic cascade through multiple mechanisms, perhaps by supporting an invasive microenvironment for both normal and tumor cells.

Autotaxin (ATX), a potent tumor motogen, augments invasive and metastatic potential of ras-transformed cells.

Autotaxin (ATX), an exo-nucleotide pyrophosphatase and phosphodiesterase, was originally isolated as a potent stimulator of tumor cell motility. In order to study whether ATX expression affects motility-dependent processes such as invasion and metastasis, we stably transfected full-length ATX cDNA into two non-expressing cell lines, parental and ras-transformed NIH3T3 (clone7) cells. The effect of ATX secretion on in vitro cell motility was variable. The ras-transformed, ATX-secreting subclones had enhanced motility to ATX as chemoattractant, but there was little difference in the motility responses of NIH3T3 cells transfected with atx, an inactive mutant gene, or empty vector. In MatrigelTM invasion assays, all subclones, which secreted enzymatically active ATX, demonstrated greater spontaneous and ATX-stimulated invasion than appropriate controls. This difference in invasiveness was not caused by differences in gelatinase production, which was constant within each group of transfectants. In vivo studies with athymic nude mice demonstrated that injection of atx-transfected NIH3T3 cells resulted in a weak tumorigenic capacity with few experimental metastases. Combination of ATX expression with ras transformation produced cells with greatly amplified tumorigenesis and metastatic potential compared to ras-transformed controls. Thus, ATX appears to augment cellular characteristics necessary for tumor aggressiveness.

The RI alpha subunit of protein kinase A controls serum dependency and entry into cell cycle of human mammary epithelial cells.

MCF-10A, a nontransformed mammary epithelial cell line, requires a mixture of hormones and growth factors for optimal cell proliferation. In this report we show that when MCF-10A cells are cultured in serum free medium they become quiescent and accumulate in G0/G1 phases of the cell cycle. Following addition of complete medium to quiescent cells, MCF-10A cells enter into the S phase within 15-18 h and resume the cell cycle distribution of proliferating cells within 24 h. Measurement of RI alpha subunit of the cAMP-dependent protein kinase (PKA) shows a 10- to 15-fold increase in protein levels at 6 h following complete medium addition, thus preceding cell entry into the S phase. Retroviral vector-mediated overexpression of RI alpha, but not of RII beta or C alpha subunits of PKA, enables MCF-10A cells to grow in serum-free medium. In addition, RI alpha downregulation by specific antisense oligodeoxynucleotide treatment or following infection with a retroviral vector containing the RI alpha cDNA in antisense orientation determines growth arrest of proliferating MCF-10A cells and is able to partially block S phase entry of quiescent MCF-10A cells following complete medium addition. These results suggest that RI alpha/PKAI is involved in the control of cell cycle progression of mammary epithelial cells at a G1 to S transition border, and that its overexpression is able to overcome serum and growth factors requirement for cell proliferation.

The regulatory subunit of cAMP-dependent protein kinase as a target for chemotherapy of cancer and other cellular dysfunctional-related diseases.

Three separate experimental approaches, using site-selective cAMP analogs, antisense strategy and retroviral vector-mediated gene transfer, have provided evidence that two isoforms, 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 RI expression is enhanced during chemical or viral carcinogenesis, in human cancer cell lines and in primary human tumors, it is a target for cancer diagnosis and therapy. 8-Cl-cAMP and RI antisense oligodeoxynucleotide, those that effectively down-regulate RI alpha and up-regulate RII beta, provide new approaches toward the treatment of cancer. This approach to modulation of RI vs RII cAMP transducers may also be beneficial toward therapy of endocrine or cellular dysfunction-related diseases where abnormal signal transduction of cAMP is critically involved.

Role of site-selective cAMP analogs in the control and reversal of malignancy.

Two isoforms of cAMP receptor protein, RI and RII, the regulatory subunits of cAMP-dependent protein kinase, transduce opposite signals, the RI being stimulatory and the RII being inhibitory of cell proliferation. In normal cells RI and RII exist at a specific physiological ratio whereas in cancer cells such physiological balance of these receptor proteins is disrupted. Reversal and suppression of malignancy can be achieved when the physiologic ratio of these intracellular signal transducers of cAMP is restored as shown by the use of site-selective cAMP analogs, antisense oligodeoxynucleotides or gene transfer, suggesting new approaches to cancer control.

Site-selective cAMP analogs induce nuclear translocation of the RII cAMP receptor protein in Ha-MuSV-transformed NIH/3T3 cells.

Site-selective cAMP analogs, depending on the position of their substituents on the adenine ring, selectively bind to either site 1 or site 2 of the known cAMP binding sites of protein kinase. Treatment of Harvey murine sarcoma virus-transformed NIH/3T3 cells with such site-selective analogs results in growth inhibition and phenotypic reversion, and the combination of a C-8 thio or halogen analog (site 1 selective) with an N6 analog (site 2 selective) produces a synergistic effect. We report here that the growth inhibitory effect of the analogs correlates with the nuclear translocation of the RII cAMP receptor protein, the regulatory subunit of protein kinase type II. The transformed NIH/3T3 cells contained no detectable level of RII in the nucleus, whereas nontransformed NIH/3T3 cells exhibited a high level of nuclear RII. Within 30 min after treatment of the transformed cells with the site-selective analogs, immunofluorescence against the RII protein markedly increased in the cell nucleus. The nuclear translocation of the RII cAMP receptor protein is an early event in the reverse transformation of the fibroblasts treated with site-selective cAMP analogs.

Site-selective 8-chloroadenosine 3',5'-cyclic monophosphate inhibits transformation and transforming growth factor alpha production in Ki-ras-transformed rat fibroblasts.

A site-selective cAMP analog, 8-chloroadenosine 3',5'-cyclic monophosphate (8-Cl-cAMP), was demonstrated to be a potent inhibitor of both the monolayer and soft agar growth of normal rat kidney (NRK) fibroblasts that had been transformed with the v-Ki-ras oncogene or treated with transforming growth factor alpha (TGF alpha). The growth inhibition was dose dependent and reversible and was accompanied by reversion of the transformed phenotype, suppression of TGF alpha production, and a decrease in p21 ras protein levels. These effects of 8-Cl-cAMP were linked to the cAMP analog's selective modulation of the type I and type II cAMP-dependent protein kinase regulatory subunits, RI and RII, present in Ki-ras-transformed and TGF alpha-treated NRK cells.

Site-selective 8-Cl-cAMP which causes growth inhibition and differentiation increases DNA (CRE)-binding activity in cancer cells.

Control mechanisms of normal differentiation are disrupted in cancer cells but can be restored by treatment with site-selective cAMP analogs. The cellular events associated with such changes entail compartmental redistribution of the cAMP-dependent protein kinase type II regulatory subunit, RII beta. The results of this study indicate that the molecular mechanisms of action involve changes in specific DNA-binding activity of putative transcription factors. Gel retardation analyses revealed that nuclear extracts from cells of various human cancer cell lines [colon cancer (LS-174T), gastric cancer (TMK-1), and leukemia (K-562)] and rodent pheochromocytoma (PC12) show a concentration-dependent increase in binding activity to a synthetic DNA that contained the cAMP-responsive element 5'-TGACGTCA-3' after treatment with 8-Cl-cAMP. Such an increase in cAMP-responsive element binding activity was not observed in the 8-C1-cAMP-unresponsive MKN-1 gastric cancer cells. These findings indicate that the antitumor activity of site-selective cAMP analogs may reside in the induction of transcription factors that restore normal gene regulation in cancer cells.