Biomedical subjects

T Clair

Publications and source records attributed to T Clair.

At least 37 records · Page 2Linked to original sources

PubMed1994

cDNA cloning of the human tumor motility-stimulating protein, autotaxin, reveals a homology with phosphodiesterases.

A human cDNA clone encoding autotaxin, a tumor cell motility-stimulating protein, reveals that this protein is an ecto/exo-enzyme with significant homology to the plasma cell membrane differentiation antigen PC-1. ATX is a 125-kDa glycoprotein, previously isolated from a human melanoma cell line (A2058), which elicits chemotactic and chemokinetic responses at picomolar to nanomolar concentrations. Affinity-purified antipeptide antibodies to the ATX peptide, ATX-102, were employed to screen an A2058 cDNA expression library made in lambda gt11. The partial cDNA sequence which was obtained was then extended by utilizing reverse transcriptase on total cellular RNA followed by polymerase chain reaction amplification. The isolated cDNA clone contained 3251 base pairs, and the mRNA message size was approximately 3.3 kilobases. The deduced amino acid sequence of autotaxin matched 30 previously sequenced peptides and comprised a protein of 915 amino acids. Data base analysis of the ATX sequence revealed a 45% amino acid identity (including 30 out of 33 cysteines) with PC-1, a pyrophosphatase/type I phosphodiesterase expressed on the surface of activated B cells and plasma cells. ATX, like PC-1, was found to hydrolyze the type I phosphodiesterase substrate p-nitrophenyl thymidine-5'-monophosphate. Autotaxin now defines a novel motility-regulating function for this class of ecto/exo-enzymes.

Amino Acid Sequence↗

PubMed1994

Flow-cytometric detection of the RI alpha subunit of type I cAMP-dependent protein kinase in human cells.

cAMP-dependent protein kinase (PKA) is composed of two genetically distinct catalytic (C) and regulatory (R) subunits. There are two different classes of PKA, designated as type I and type II, which contain distinct R subunits (RI or RII, respectively) but share a common C subunit. Enhanced expression of type I PKA has been correlated with cell proliferation and neoplastic transformation. Detection of the different PKA subunits is usually performed by photoaffinity labeling with 8-N3-32P-cAMP or by radioimmunolabeling techniques. Both techniques are time consuming and require a high number of cells and the use of radioactive reagents. Using the MCF-10A normal human mammary cell line infected with a recombinant retroviral vector containing the human RI alpha gene (MCF-10A RI alpha), we have developed a flow-cytometric assay to detect the intracellular content of RI alpha protein in human cells. MCF-10A and MCF-10A RI alpha cells were fixed in 1.5% paraformaldehyde at 37 degrees C for 15 min and permeabilized by methanol and acetone (1:1) at -20 degrees C for 5 min before staining with a specific IgG2a MoAb followed by a FITC-conjugate rabbit-anti mouse IgG. This procedure was also successfully utilized to recognize RI alpha protein content in human peripheral blood lymphocytes. Flow-cytometric detection of the RI alpha subunit in human cells is feasible and allows the study of the role of type I PKA in cell growth and neoplastic transformation.

Breast↗

PubMed1994

Protein kinase A (PK-A) regulatory subunit expression in colorectal cancer and related mucosa.

Photoaffinity labelling (PAL) with [32P]8-azido-cAMP and polyacrylamide gel electrophoresis (PAGE) has been used to identify three specific cAMP-binding proteins (cAMP-BPs) within cytosols derived from the centre and periphery of 32 human colorectal cancers and from related adjacent (less than 5 cm from the tumour) and distant (more than 5 cm from the tumour) microscopically benign mucosa. By immunoprecipitation with specific anti-RI and anti-RII antibodies these proteins have subsequently been characterised as a single form of RI (48 kDa) and two forms of RII (50 and 52 kDa). The relative expression of isoforms in each specimen has been quantified by laser densitometry. There was significantly more RI expressed in both tumour centre and periphery than in either adjacent or distant mucosa (P < 0.008 by Wilcoxon signed-rank test). There was no significant difference in relative RI expression between tumour centre and periphery, or between adjacent and distant mucosa. There was no association between relative RI expression and Dukes' stage. Poorly differentiated tumours expressed significantly more RI than those that were either moderately or well differentiated (P = 0.016 by Mann-Whitney U-test). This study is the first to have characterised cAMP-BPs within human colorectal tissues and has demonstrated that colorectal cancers, and in particular those of poor histological grade, relatively overexpress RI when compared with related benign mucosa.

Affinity Labels↗

PubMed1994

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.

Animals↗

PubMed1994

Retroviral vector-mediated overexpression of the RII beta subunit of the cAMP-dependent protein kinase induces differentiation in human leukemia cells and reverts the transformed phenotype of mouse fibroblasts.

We have recently shown, using antisense strategy, that the RII beta regulatory subunit of cAMP-dependent protein kinase is essential for cAMP-induced growth inhibition and differentiation of HL-60 human leukemia cells. We constructed a retroviral vector for RII beta (MT-RII beta) by inserting human RII beta complementary DNA into the OT1521 retroviral vector plasmid that contains an internal mouse metallothionein-1 promoter and a neomycin resistance gene. The PA317 packaging cell line was then transfected with MT-RII beta plasmid to produce the amphotrophic stock of MT-RII beta retroviral vector. The infection with MT-RII beta and treatment with CdCl2 brought about growth arrest in HL-60 human leukemia and Ki-ras-transformed NIH 3T3 clone DT cells in monolayer culture with no sign of toxicity. The growth inhibition correlated with the expression of RII beta and accompanied changes in cell morphology; cells became flat, exhibiting enlarged cytoplasm. The growth of these cells in semisolid medium (anchorage-independent growth) was almost completely suppressed. In contrast, overexpression of the RI alpha subunit of protein kinase enhanced the cell proliferation in DT cells. The MT-RII beta-infected cells exhibited an increased sensitivity toward treatment with cAMP analogues, such as 8-Cl-cAMP and N6-benzyl-cAMP, as compared with the parental noninfected cells. In MT-RII beta HL-60 cells, N6-benzyl-cAMP treatment greatly enhanced the expression of monocytic surface markers. These results suggest that the RII beta cAMP receptor, by binding to its ligand, cAMP, acts as a tumor suppressor protein exerting growth inhibition, differentiation, and reverse transformation.

3T3 Cells↗

PubMed1993

8-Cl-cAMP induces truncation and down-regulation of the RI alpha subunit and up-regulation of the RII beta subunit of cAMP-dependent protein kinase leading to type II holoenzyme-dependent growth inhibition and differentiation of HL-60 leukemia cells.

8-Cl-cAMP, a site-selective cAMP analog, induces growth inhibition in a variety of cell types of human cancer cell lines. This inhibitory effect of 8-Cl-cAMP was related to its ability to differentially regulate type I versus type II cAMP-dependent protein kinase. In the present study we demonstrated a unique mechanism of action of 8-Cl-cAMP in the regulation of these kinase isozymes in HL-60 human promyelocytic leukemia cells. High-performance liquid chromatography (HPLC) resolved various isoforms of protein kinase present in HL-60 cells. In control cells, type I protein kinase (PKI) comprised more than 90% and type II protein kinase (PKII) less than 10% of the total cAMP-stimulated kinase activity. Treatment with 8-Cl-cAMP (5 microM, 72 h) decreased PKI to a level below 30% of that in untreated control cells and markedly increased PKII composed of three peaks. Photoaffinity labeling/SDS-polyacrylamide gel electrophoresis of column fractions identified the molecular species of regulatory (R) subunits present in protein kinases. Control cells contained high levels of the 48-kDa protein (RI) that composed PKI and low levels of the 50-kDa RII associated with PKII. 8-Cl-cAMP treatment brought about a decrease in the 48-kDa RI along with an increased formation of the truncated 34-kDa RI associated with PKI and an increase in the 50-54-kDa species of RII associated with PKII. A similar protein kinase profile as that shown by 8-Cl-cAMP treatment was observed in cells infected with the human RII beta retroviral vector: the 48-kDa RI of PKI decreased and the 52- and 54-kDa RII associated with PKII increased as compared with uninfected control cells. However, unlike 8-Cl-cAMP treatment, RII beta retroviral vector infection brought about no increase in the 34-kDa-truncated RI but exhibited an increase in the free 48-kDa RI subunit. As the 48-kDa RI and the 50-kDa RII were present in control cells, the enhanced expression of the 52- and 54-kDa RII proteins was due to overexpression of the RII beta gene. We identified the 48-kDa RI as RI alpha, the 50-kDa RII as RII alpha, the 52-kDa RII as RII beta, and the 54-kDa RII as the phosphorylated form of either the RII alpha or RII beta subunit. In vivo labeling experiments using [3H]8-Cl-cAMP demonstrated that 8-Cl-cAMP enters cells and binds to both PKI and PKII.(ABSTRACT TRUNCATED AT 400 WORDS)

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1993

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.

Amino Acid Sequence↗

PubMed1993

Thyroid-stimulating hormone-regulated growth and cell cycle distribution of thyroid cells involve type I isozyme of cyclic AMP-dependent protein kinase.

Optimal growth and differentiation of normal rat thyroid FRTL5 cells depend strictly on the presence of thyroid-stimulating hormone (TSH). FRTL5 cells deprived of TSH cease dividing and become quiescent. Addition of TSH to quiescent cells, which activates the cyclic AMP-mediated pathway, is sufficient to stimulate cell entry into S phase of the cell cycle. We have previously shown that the differential expression of the two isozymes, type I and type II, of the cyclic AMP-dependent protein kinase (PKA) correlates with cell growth and differentiation of several rodent and human cell lines. We have studied the role of PKA in the TSH-regulated growth and cell cycle distribution of FRTL5 cells. Upon addition of TSH to FRTL5 cells deprived of hormone, a rapid induction of RI alpha mRNA species occurred within 30 min after treatment, reaching the levels of proliferating FRTL5 cells at 12 h. RII alpha mRNA levels slightly increased after TSH addition, whereas C alpha mRNA levels did not show major changes. Photoaffinity labeling of PKA receptor proteins showed that addition of TSH to quiescent FRTL5 cells induced a progressive increase in RI alpha levels starting at 6 h after stimulation, whereas RII alpha receptor levels increased only slightly. When FRTL5 cells were treated with an antisense oligodeoxynucleotide targeted against the RI alpha regulatory subunit, their growth was arrested, whereas an antisense against the RII alpha regulatory subunit produced only a mild growth inhibition. Moreover, exposure to the antisense RI alpha oligomer resulted in accumulation of cells in the G0-G1 compartment, as during TSH deprivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Carcinoembryonic antigen regulation in human colorectal tumor cells by a site-selective cyclic AMP analogue: a comparison with interferon-gamma.

Treatment of human colorectal tumor cells (LS174T, HT-29, and WiDr) with analogues of cyclic AMP (cAMP) (dibutyryl-cAMP and 8-Cl-cAMP) selectively enhances the expression of carcinoembryonic antigen (CEA). Dose and temporal kinetics results revealed that 8-Cl-cAMP was approximately 100-fold more potent than dibutyryl-cAMP for increasing CEA expression. Results demonstrated that 8-Cl-cAMP treatment of LS174T quantitatively increased CEA levels in cell extracts 2-fold, increased anti-CEA monoclonal antibody (MAb) binding to the tumor cell surface, and induced the appearance of CEA-related mRNA transcripts. The findings suggest that 8-Cl-cAMP is capable of regulating CEA expression at transcriptional and/or post-transcriptional levels. Other human tumor cells, as well as normal cell types which do not constitutively express CEA, remained CEA-negative following 8-Cl-cAMP treatment. Moreover, the level of expression of other human tumor antigens as well as antigens of the major histocompatibility complex were not changed by 8-Cl-cAMP treatment, suggesting some selectivity for CEA regulation by this cAMP analogue. In vivo administration of 8-Cl-cAMP to athymic mice bearing LS174T tumor xenografts increased the amount of anti-CEA MAb bound to tumor extracts as well as the tumor localization of a radionuclide-conjugated anti-CEA MAb. The results indicate that 8-Cl-cAMP can selectively upregulate CEA expression on human colorectal tumor cells in vitro and in vivo. Interestingly, IFN-gamma treatment of the LS174T cells fails to enhance or induce expression of CEA or any of the histocompatibility leukocyte antigens. Thus, 8-Cl-cAMP treatment regulates CEA expression through another cellular pathway which may involve cAMP-dependent protein kinase.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1991

Differentiation of HL-60 leukemia by type I regulatory subunit antisense oligodeoxynucleotide of cAMP-dependent protein kinase.

A marked decrease in the type I cAMP-dependent protein kinase regulatory subunit (RI alpha) and an increase in the type II protein kinase regulatory subunit (RII beta) correlate with growth inhibition and differentiation induced in a variety of types of human cancer cells, in vitro and in vivo, by site-selective cAMP analogs. To directly determine whether RI alpha is a growth-inducing protein essential for neoplastic cell growth, human HL-60 promyelocytic leukemia cells were exposed to 21-mer RI alpha antisense oligodeoxynucleotide, and the effects on cell replication and differentiation were examined. The RI alpha antisense oligomer brought about growth inhibition and monocytic differentiation, bypassing the effects of an exogenous cAMP analog. These effects of RI alpha antisense oligodeoxynucleotide correlated with a decrease in RI alpha receptor and an increase in RII beta receptor level. The growth inhibition and differentiation were abolished, however, when these cells were exposed simultaneously to both RI alpha and RII beta antisense oligodeoxynucleotides. The RII beta antisense oligodeoxynucleotide alone has been previously shown to specifically block the differentiation inducible by cAMP analogs. These results provide direct evidence that RI alpha cAMP receptor plays a critical role in neoplastic cell growth and that cAMP receptor isoforms display specific roles in cAMP regulation of cell growth and differentiation.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1991

Suppression of malignancy targeting the intracellular signal transducing proteins of cAMP: the use of site-selective cAMP analogs, antisense strategy, and gene transfer.

An hypothesis has been presented suggesting that two isoforms of cAMP receptor proteins are crucial effectors in tumorigenesis. The evidence in support of this hypothesis shows that: (1) cAMP transduces dual controls, both positive and negative, on cell growth and differentiation. (2) Such dual controls are respectively governed by two isoforms of cAMP receptor proteins, the type I and type II regulatory subunits of cAMP-dependent protein kinase. (3) In normal physiology, the functional balance of these cAMP receptor isoforms is strictly controlled to meet either stimulation or inhibition of cell growth as it is required, whereas such control is lost in cancer cells. (4) Cancer cells can also be made to differentiate and acquire growth control when the functional balance of these intracellular signal transducers of cAMP is restored by the use of site-selective cAMP analogs, antisense strategy, or gene transfer, suggesting new approaches to cancer therapy.

Animals↗

PubMed1991

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.

Animals↗

PubMed1990

8-Chloro-cAMP inhibits transforming growth factor alpha transformation of mammary epithelial cells by restoration of the normal mRNA patterns for cAMP-dependent protein kinase regulatory subunit isoforms which show disruption upon transformation.

Differential regulation of the regulatory subunits of cAMP-dependent protein kinase isozymes correlates with the growth inhibitory effect of site-selective 8-Cl-cAMP demonstrated in cancer cell lines (Ally, S., Tortora, G., Clair, T., Grieco, D., Merlo, G., Katsaros, D., Ogreid, D., Døskeland, S.O., Jahnsen, T., and Cho-Chung, Y.S. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6319-6322). Such selective modulation of protein kinase isozyme regulatory subunits was also found in the 8-Cl-cAMP-induced inhibition of both transformation and transforming growth factor alpha (TGF alpha) production in Ki-ras-transformed rat kidney fibroblasts (Tortora, G., Ciardiello, F., Ally, S., Clair, T., Salomon, D. S., and Cho-Chung, Y. S. (1989) FEBS Lett. 242, 363-367). In this work, we have demonstrated that 8-Cl-cAMP antagonizes the TGF alpha effect in TGF alpha-transformed mouse mammary epithelial cells (NOG-8TFC17) at the level of gene expression for cAMP receptor protein isoforms, RI and RII (the regulatory subunits of protein kinase isozymes). Northern blot analysis demonstrated that in the transformed NOG-8TFC17 cells, compared with the nontransformed counterpart NOG-8 cells, the mRNA levels for the RI alpha cAMP receptor protein markedly increased, whereas the mRNA levels for the RII alpha and RII beta cAMP receptor proteins decreased. 8-Cl-cAMP, which induced growth inhibition and phenotypic reversion in NOG-8TFC17 cells, caused an inverse change in the mRNA patterns of the cAMP receptor proteins; RI alpha cAMP receptor mRNA sharply decreased to levels comparable with that of the nontransformed NOG-8 cells, whereas RII beta mRNA increased to a level even greater than that in the NOG-8 cells. In addition, one mRNA species of RII alpha increased, whereas the other RII alpha mRNA species decreased during the treatment. The mRNA level for the catalytic subunit of protein kinase, however, did not change during 8-Cl-cAMP treatment. In addition, 8-Cl-cAMP brought about a reduction in both TGF alpha mRNA and protein levels. These coordinated changes in the expression of the cAMP receptor proteins and TGF alpha were not observed during cis-hydroxyprolineor TGF beta-induced growth inhibition of the NOG-8TFC17 cells. Thus, the antagonistic effect of 8-Cl-cAMP toward TGF alpha-induced transformation involves modulation of the expression of a specific set of cellular genes.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1990

An antisense oligodeoxynucleotide targeted against the type II beta regulatory subunit mRNA of protein kinase inhibits cAMP-induced differentiation in HL-60 leukemia cells without affecting phorbol ester effects.

The type II beta regulatory subunit of cAMP-dependent protein kinase (RII beta) has been hypothesized to play an important role in the growth inhibition and differentiation induced by site-selective cAMP analogs in human cancer cells, but direct proof of this function has been lacking. To address this issue, HL-60 human promyelocytic leukemia cells were exposed to RII beta antisense synthetic oligodeoxynucleotide, and the effects on cAMP-induced growth regulation were examined. Exposure of these cells to RII beta antisense oligodeoxynucleotide resulted in a decrease in cAMP analog-induced growth inhibition and differentiation without apparent effect on differentiation induced by phorbol esters. This loss in cAMP growth regulatory function correlated with a decrease in basal and induced levels of RII beta protein. Exposure to RII beta sense, RI alpha and RII alpha antisense, or irrelevant oligodeoxynucleotides had no such effect. These results show that the RII beta regulatory subunit of protein kinase plays a critical role in the cAMP-induced growth regulation of HL-60 leukemia cells.

Base Sequence↗

PubMed1989

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.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1989

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.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1989

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.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1989

Induction of megakaryocytic differentiation and modulation of protein kinase gene expression by site-selective cAMP analogs in K-562 human leukemic cells.

Two classes (site 1- and site 2-selective) of cAMP analogs, which either alone or in combination demonstrate a preference for binding to type II rather than type I cAMP-dependent protein kinase isozyme, potently inhibit growth in a spectrum of human cancer cell lines in culture. Treatment of K-562 human leukemic cells for 3 days with 30 and 10 microM 8-chloroadenosine 3',5'-cyclic monophosphate (8-Cl-cAMP) (site 1-selective) resulted in 60% and 20% growth inhibition, respectively (with over 90% viability). N6-Benzyl-cAMP (site 2-selective) (30 microM) treatment resulted in 20% growth inhibition by day 3. When 8-Cl-cAMP (10 microM) and N6-benzyl-cAMP (30 microM) were both added, growth was almost completely arrested. The growth inhibition was accompanied by megakaryocytic differentiation in K-562 cells. The untreated control cells expressed little or no detectable levels of glycoprotein IIb-IIIa surface antigen complex. 8-Cl-cAMP (30 microM) treatment for 3 days substantially increased the antigen expression, while N6-benzyl-cAMP caused little or no change in the antigen expression. When cells were treated with 8-Cl-cAMP in combination with N6-benzyl-cAMP, antigen expression was synergistically enhanced, and cells demonstrated megakaryocyte morphology. By Northern blotting, we examined the mRNA levels of the type I and type II protein kinase regulatory subunits (RI alpha and RII beta), the catalytic subunit, and c-myc during 8-Cl-cAMP treatment. The steady-state level of RII beta cAMP receptor mRNA sharply increased within 1 hr of treatment and remained elevated for 3 days, while that of the RI alpha receptor markedly decreased to below control level within 6 hr and remained low during treatment. However, 8-Cl-cAMP did not affect the mRNA level of the catalytic subunit. 8-Cl-cAMP treatment also brought about a rapid decrease in c-myc mRNA. Thus, differential regulation of cAMP receptor genes is an early event in cAMP-induced differentiation and growth control of K-562 leukemia cells.

Antigens, Surface↗