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.     

Activation and interaction with protein kinase C of a cytoplasmic tyrosine kinase, Itk/Tsk/Emt, on Fc epsilon RI cross-linking on mast cells

Cross-linking of the high-affinity IgE receptor (Fc epsilon RI) on mast cells induces rapid phosphorylation on serine, threonine, and tyrosine residues and increases the enzymatic activity, of a Tec subfamily tyrosine kinase, Itk/Tsk/Emt (Emt). The pleckstrin homology domain of Emt at its amino-terminal interacts directly with multiple isoforms of protein kinase C (PKC) in vitro. In addition, a portion of Emt is physically associated with multiple isoforms of PKC in intact mast cells. PKC phosphorylates a bacterial fusion protein containing the pleckstrin homology domain of Emt in vitro. Coexpression of Emt in COS-7 cells with Ca(2+)-dependent PKC isoforms (alpha, beta I, or beta II) induces an enhancement in tyrosine phosphorylation of Emt. In vivo inhibition of PKC expression or activity attenuates tyrosine phosphorylation and enzymatic activity of Emt induced upon Fc epsilon RI cross-linking. These data collectively suggest that PKC phosphorylates Emt and activates its autophosphorylating activity. Alternatively, PKC could activate another tyrosine kinase that phosphorylates Emt, or PKC-mediated phosphorylation of Emt may render it a target for another tyrosine kinase. In any case, PKC appears to play a major role in the activation of Emt induced upon Fc epsilon RI cross-linking.     

Presence of IgM antibodies to Trypanosoma cruzi urinary antigen in sera from patients with acute Chagas'' disease

We have investigated the ability of an antisense immunoglobulin E (IgE) receptor alpha-subunit oligodeoxynucleotide (Fc epsilon RI alpha ODN) specifically to inhibit IgE-mediated allergic reactions in the mouse. Synthetic antisense Fc epsilon RI alpha ODN dose-dependently inhibited passive cutaneous anaphylaxis and histamine release from the mouse peritoneal mast cells (MPMC) activated by anti-dinitrophenyl (DNP) IgE. Northern blot analysis showed that the mast cells treated with antisense Fc epsilon RI alpha ODN exhibited no detectable levels of L-histidine decarboxylase mRNA after anti-DNP IgE stimulation, whereas the cells treated with sense Fc epsilon RI alpha ODN possessed significant amounts of this mRNA. Examination of the elevation of cAMP levels in MPMC following the activation with anti-DNP IgE demonstrated a significant rise in activated cells, but not in the antisense Fc epsilon RI alpha ODN-treated cells. Moreover, antisense Fc epsilon RI alpha ODN had a significant inhibitory effect on anti-DNP IgE-induced tumour necrosis factor-alpha production. Our results demonstrated that antisense Fc epsilon RI alpha ODN inhibited the IgE-mediated allergic reaction in vivo and in vitro.     

Exposure to duty-related incident stressors in urban firefighters and paramedics

Peptide growth factors regulate normal cellular proliferation and differentiation through autocrine and paracrine pathways and are involved in cancer development and progression. Among the endogenous growth factors, the epidermal growth factor (EGF)-related proteins play an important role in the pathogenesis of human cancer. In fact, overexpression of EGF-related growth factors such as transforming growth factor alpha and amphiregulin and/or their specific receptor, the EGF receptor (EGFR), has been detected in several types of human cancers, including breast, lung, and colorectal cancers. Therefore, the blockade of EGFR activation by using anti-EGFR monoclonal antibodies (MAbs) has been proposed as a potential anticancer therapy. The cAMP-dependent protein kinase (PKA) is an intracellular enzyme with serine-threonine kinase activity that plays a key role in cell growth and differentiation. Two PKA isoforms with identical catalytic (C) subunits but different cAMP-binding regulatory (R) subunits (defined as RI in PKAI and RII in PKAII) have been identified. Predominant expression of PKAII is found in normal nonproliferating tissues and in growth-arrested cells, whereas enhanced levels of PKAI are detected steadily in tumor cells and transiently in normal cells exposed to mitogenic stimuli. Overexpression of PKAI has been correlated recently with poor prognosis in breast cancer patients. Inhibition of PKAI expression and function by specific pharmacological agents such as the selective cAMP analogue 8-chloro-cAMP (8-Cl-cAMP) induces growth inhibition in various human cancer cell lines in vitro and in vivo. We have provided experimental evidence of a functional cross-talk between ligand-induced EGFR activation and PKAI expression and function. In fact, PKAI is overexpressed and activated following transforming growth factor alpha-induced transformation in several rodent and human cell line models. Furthermore, PKAI is involved in the intracellular mitogenic signaling following ligand-induced EGFR activation. We have shown that an interaction between EGFR and PKAI occurs through direct binding of the RI subunit to the Grb2 adaptor protein. In this respect, PKAI seems to function downstream of the EGFR, and experimental evidence suggests that PKAI is acting upstream of the mitogen-activated protein kinase pathway. We have also demonstrated that the functional interaction between the EGFR and the PKAI pathways could have potential therapeutic implications. In fact, the combined interference with both EGFR and PKAI with specific pharmacological agents, such as anti-EGFR blocking MAbs and cAMP analogues, has a cooperative antiproliferative effect on human cancer cell lines in vitro and in vivo. The antitumor activity of this combination could be explored in a clinical setting because both the 8-Cl-cAMP analogue and the anti-EGFR blocking MAb C225 have entered human clinical trial evaluation. Finally, both MAb C225 and 8-Cl-cAMP are specific inhibitors of intracellular mitogenic signaling that have different mechanisms of action compared with conventional cytotoxic drugs. In this respect, a cooperative growth-inhibitory effect in combination with several chemotherapeutic agents in a large series of human cancer cell lines in vitro and in vivo has been demonstrated for anti-EGFR blocking MAbs or for 8-Cl-cAMP. Therefore, the combination of MAb C225 and 8-Cl-cAMP following chemotherapy could be investigated in cancer patients.     

Differentiation and transforming growth factor-beta receptor down-regulation by collagen-alpha2beta1 integrin interaction is mediated by focal adhesion kinase and its downstream signals in murine osteoblastic cells

Interaction of type I collagen (COL(I)) with alpha2beta1 integrin causes differentiation and transforming growth factor (TGF)-beta receptor down-regulation in osteoblastic cells (Takeuchi, Y., Nakayama, K., and Matsumoto, T. (1996) J. Biol. Chem. 271, 3938-3644). The TGF-beta receptor down-regulation enables cells to escape from the inhibition of differentiation by TGF-beta. To clarify how the cell-matrix interaction regulates these phenotypic changes, signaling pathways were examined in murine MC3T3-E1 cells. Attachment of cells to COL(I) stimulated tyrosine phosphorylation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK), a mitogen-activated protein kinase (MAPK), and enhanced MAPK activity. Inhibition of tyrosine kinase by herbimycin A, destruction of focal adhesion by cytochalasin D, or overexpression of antisense FAK mRNA prevented the activation of ERK/MAPK and the increase in alkaline phosphatase (ALP) activity. Transient expression of a MAPK-specific phosphatase, CL100, also suppressed the elevation of ALP activity. In addition, introduction of a constitutively active MAPK kinase enhanced ALP activity in the absence of collagen production. TGF-beta receptor down-regulation was abrogated by treatments that inactivate FAK, whereas the expression of CL100 had no effect. These results demonstrate that COL(I)-alpha2beta1 integrin interaction facilitates differentiation and down-regulates TGF-beta receptors via the activation of FAK and its diverse downstream signals. These signaling pathways may play an important role in the sequential differentiation of osteoblasts during bone formation.     

Treatment of glioblastoma U-87 by systemic administration of an antisense protein kinase C-alpha phosphorothioate oligodeoxynucleotide

Glioblastoma multiforme is the most common form of malignant brain cancer in adults and, unfortunately, is not amenable to treatment with current therapeutic modalities. Human glioblastoma U-87 has many of the distinguishing phenotypic features of primary glioblastoma, including an autocrine form of proliferation, high levels of protein kinase C alpha (PKC alpha), and infiltration via white matter tracts. We show that treatment of mice bearing U-87 xenografts with an antisense phosphorothioate oligodeoxynucleotide (S-oligodeoxynucleotide) against the 3'-untranslated region of PKC alpha mRNA results in suppression of tumor growth. Growth was inhibited in both subcutaneous and intracranial tumors, and in the latter instance, treatment with the antisense PKC alpha S-oligodeoxynucleotide resulted in a doubling in median survival time ( > 80 days), with 40% long term survivors. The antisense S-oligodeoxynucleotide did not produce systemic toxicity in mice with subcutaneous or intracranial tumors after daily intraperitoneal injection for 21 or 80 days, respectively, and a scrambled S-oligodeoxynucleotide with the same nucleotide composition as the antisense S-oligodeoxynucleotide did not produce an antitumor effect. The intratumoral levels of both antisense and scrambled S-oligodeoxynucleotide in subcutaneous tumors were 2 microM after 21 daily doses of 20 mg/kg S-oligodeoxynucleotide. The antisense S-oligodeoxynucleotide selectively reduced the levels of PKC alpha in subcutaneous tumors but not those of protein kinase C epsilon or protein kinase C zeta. This is the first demonstration that the growth of glioblastoma multiforme can be suppressed by an antisense PKC alpha S-oligodeoxynucleotide and suggests that this may represent an effective therapy for this type of malignancy.     

Targeted downregulation of caveolin-1 is sufficient to drive cell transformation and hyperactivate the p42/44 MAP kinase cascade

Caveolin-1 is a principal component of caveolae membranes in vivo. Caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes. Interestingly, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (7q31.1). However, it remains unknown whether downregulation of caveolin-1 is sufficient to mediate cell transformation or tumorigenicity. Here, we employ an antisense approach to derive stable NIH 3T3 cell lines that express dramatically reduced levels of caveolin-1 but contain normal amounts of caveolin-2. NIH 3T3 cells harboring antisense caveolin-1 exhibit anchorage-independent growth, form tumors in immunodeficient mice and show hyperactivation of the p42/44 MAP kinase cascade. Importantly, transformation induced by caveolin-1 downregulation is reversed when caveolin-1 protein levels are restored to normal by loss of the caveolin-1 antisense vector. In addition, we show that in normal NIH 3T3 cells, caveolin-1 expression levels are tightly regulated by specific growth factor stimuli and cell density. Our results suggest that upregulation of caveolin-1 may be important in mediating contact inhibition and negatively regulating the activation state of the p42/44 MAP kinase cascade.     

Expression of the antisense cDNA for protein kinase C alpha attenuates resistance in doxorubicin-resistant MCF-7 breast carcinoma cells

Multidrug resistance is functionally associated with the expression of a plasma membrane energy-dependent drug efflux pump termed P-glycoprotein, the product of the mdr1 gene. Transfection of P-glycoprotein-expressing doxorubicin-resistant MCF-7 cells with an expression vector containing the cDNA for protein kinase C alpha in the antisense orientation reduces protein kinase C alpha levels and decreases total protein kinase C activity by 75%. This is accompanied by reduced phosphorylation of P-glycoprotein, a 2-fold increase in drug retention, and a 3-fold increase in doxorubicin cytotoxicity. These results provide further evidence that protein kinase C alpha can positively regulate multidrug resistance in MCF-7 cells through posttranslational phosphorylation of P-glycoprotein.     

Antiproliferative effect of DNA polymerase alpha antisense oligodeoxynucleotides on breast cancer cells

Antisense oligonucleotides appear to offer considerable promise as sequence-specific inhibitors of gene expression. Different cellular targets for oligodeoxynucleotides with oncologic interest have been identified such as oncogenes, growth factors, and cell cycle-related genes. DNA polymerase alpha (pol alpha) plays a relevant role in DNA synthesis and cell proliferation. Pol alpha gene expression is constitutive throughout the cell cycle and its mRNA content and activity are related to the growth rate and neoplastic phenotype. The effects of a 18-mer pol alpha antisense oligomer on the proliferation of the MDA-MB 231 breast cancer cell line have been investigated. After 48 h in culture with oligomers (10 microM), about 50% growth inhibition was observed in antisense-treated cells, as evaluated by 3-(4,5-dimethythiazol-2yl)-2,5-diphenyltetrazolium bromide assay and cell count. [3H]Thymidine incorporation exhibited a 90% inhibition of DNA synthesis associated to 64% accumulation of cells at the G1-S border of the cycle as by flow cytometry, at 24 h. Northern hybridization and SDS-PAGE of immunoprecipitated MDA-MB 231 cell lysates revealed a decreased expression of pol alpha mRNA and a reduction of the 180-kDa polypeptide, respectively. Collectively, the data further confirm the relevance of pol alpha in the replicative cycle, as well as strengthen the potentiality of the antisense strategy for the control of gene expression and cell growth.     

Human regulatory subunit RI beta of cAMP-dependent protein kinases: expression, holoenzyme formation and microinjection into living cells

The human regulatory subunit RI beta of cAMP-dependent protein kinases was expressed in Escherichia coli as a fusion protein with glutathione S-transferase. Purification was performed by affinity chromatography on glutathione-agarose beads after cleavage with thrombin. The human recombinant RI beta protein migrated at 55 kDa on SDS-PAGE and displayed immunoreactivity with an anti-human RI beta antiserum. Furthermore, the purified recombinant RI beta protein was shown to exist as a dimer that was able to form holoenzyme with the catalytic subunit C alpha. The rate of RI beta 2C alpha 2 holoenzyme formation was faster in the presence than in the absence of MgATP. The kinase activity measured before and after adding cAMP to the holoenzyme showed that the presence of cAMP resulted in holoenzyme dissociation and release of active C alpha-subunit, due to cAMP binding to RI beta. Compared to a RI alpha 2C alpha 2 holoenzyme, the RI beta 2C alpha 2 holoenzyme exhibited a more than twofold higher sensitivity to cAMP. The subcellular localization of RI beta was analyzed in quiescent REF-52 fibroblasts and Wistar rat thyroid (WRT) cells after microinjection of fluorescently labeled proteins into the cytoplasm. A cytoplasmic distribution was observed when free RI beta was injected, whereas free C alpha injected into the cytoplasm appeared in the nucleus. When holoenzymes with labeled RI beta and unlabeled C alpha, or unlabeled RI beta and labeled C alpha, were injected, unstimulated cells showed fluorescence in the cytoplasm of both cell types. REF-52 cells stimulated with 8-bromo-cAMP (8-Br-cAMP) and WRT cells treated with thyrotropin (TSH) showed fluorescence mainly in the cytoplasm when RI beta was the labeled subunit of the in vivo dissociated holoenzyme. In contrast, nuclear fluorescence was evident from the release and translocation of labeled C alpha from the holoenzyme complex after stimulation with 8-Br-cAMP or TSH.     

p27(kip1) acts as a downstream effector of and is coexpressed with the beta1C integrin in prostatic adenocarcinoma

Integrins are a large family of transmembrane receptors that, in addition to mediating cell adhesion, modulate cell proliferation. The beta1C integrin is an alternatively spliced variant of the beta1 subfamily that contains a unique 48-amino acid sequence in its cytoplasmic domain. We have shown previously that in vitro beta1C inhibits cell proliferation and that in vivo beta1C is expressed in nonproliferative, differentiated epithelium and is selectively downregulated in prostatic adenocarcinoma. Here we show, by immunohistochemistry and immunoblotting analysis, that beta1C is coexpressed in human prostate epithelial cells with the cell-cycle inhibitor p27(kip1), the loss of which correlates with poor prognosis in prostate cancer. In the 37 specimens analyzed, beta1C and p27(kip1) are concurrently expressed in 93% of benign and 84%-91% of tumor prostate cells. Forced expression of beta1C in vitro is accompanied by an increase in p27(kip1) levels, by inhibition of cyclin A-dependent kinase activity, and by increased association of p27(kip1) with cyclin A. beta1C inhibitory effect on cell proliferation is completely prevented by p27(kip1) antisense, but not mismatch oligonucleotides. beta1C expression does not affect either cyclin A or E levels, or cyclin E-associated kinase activity, nor the mitogen-activated protein (MAP) kinase pathway. These findings show a unique mechanism of cell growth inhibition by integrins and point to beta1C as an upstream regulator of p27(kip1) expression and, therefore, a potential target for tumor suppression in prostate cancer.     

Protein kinase C alpha expression is required for heparin inhibition of rat smooth muscle cell proliferation in vitro and in vivo

Heparin is a complex glycosaminoglycan that inhibits vascular smooth muscle cell (SMC) growth in vitro and in vivo. To define the mechanism by which heparin exerts its antiproliferative effects, we asked whether heparin interferes with the activity of intracellular protein kinase C (PKC). The membrane-associated intracellular PKC activity increased following stimulation of cultured rat SMCs with fetal calf serum and was suppressed by heparin in a time- and dose-dependent manner. Heparin acted through a selective inhibition of the PKC-alpha since preincubation of the cells with a 20-mer phosphorothioate PKC-alpha antisense oligodeoxynucleotide (ODN) eliminated the heparin effect. In vivo, following balloon injury of the rat carotid artery, particulate fraction PKC content increased with a time course and to an extent comparable with the observed changes in vitro. Heparin, administered at the time of injury or shortly thereafter, inhibited the activity of the particulate PKC and suppressed the in situ phosphorylation of an 80-kDa myristoylated alanine-rich protein kinase C substrate (MARCKS), a substrate of PKC. The topical application of the phosphorothioate antisense ODN selectively suppressed the expression of the PKC-alpha isoenzyme in vivo but did not affect injury-induced myointimal proliferation. Topical application of the ODN also eliminated the antiproliferative activity of heparin. These results therefore suggest that heparin might block SMC proliferation by interfering with the PKC pathway through a selective direct inhibition of the PKC-alpha isoenzyme.     

Inhibition of Gal beta1, 4GlcNAc alpha2,6 sialyltransferase expression by antisense-oligodeoxynucleotides

Treatment of human colorectal carcinoma cells HT29 with specific antisense oligodeoxynucleotides led to a decreased Gal beta1,4GlcNAc alpha2,6 sialyltransferase activity on the level of protein expression as well as on the mRNA level. Antisense treatment did not effect cell viability or cell growth. Oligodeoxynucleotides which were complementary to the region upstream of the initiation codon were particularly effective in inhibition of enzyme expression. No such inhibition was found by treatment of cells with oligodeoxynucleotides complementary to the region downstream of the initiation codon or by treatment of cells with scrambled controls or sense oligodeoxynucleotides.     

Effect of cisplatin and c-myb antisense phosphorothioate oligodeoxynucleotides combination on a human colon carcinoma cell line in vitro and in vivo

We investigated the effect of c-myb antisense phosphorothioate oligodeoxynucleotides [(S)ODNs] and cisplatin (CDDP) combination on the human colon carcinoma cell line LoVo Dx both in vitro and in nude mice bearing LoVo Dx solid tumour. We show that antisense (S)ODN treatment decreases c-myb mRNA and protein expression, induces growth arrest in the G1 phase of the cell cycle, and inhibits cell proliferation. In vivo treatment with c-myb antisense (S)ODNs results in a reduction in tumour growth. A greater inhibition of cell proliferation in vitro and a higher increase of tumour growth inhibition and growth delay in vivo were obtained with the combination of (S)ODNs and CDDP than when the two agents were administered separately. This comparative study, using the same tumour cell line in vitro and in vivo, suggests that c-myb antisense (S)ODNs might be useful in the therapy of colon cancer in combination with antineoplastic drugs.     

Decreased expression of BRCA1 accelerates growth and is often present during sporadic breast cancer progression

We have characterized expression of the familial breast and ovarian cancer gene, BRCA1, in cases of non-hereditary (sporadic) breast cancer and analyzed the effect of antisense inhibition of BRCA1 on the proliferative rate of mammary epithelial cells. BRCA1 mRNA levels are markedly decreased during the transition from carcinoma in situ to invasive cancer. Experimental inhibition of BRCA1 expression with antisense oligonucleotides produced accelerated growth of normal and malignant mammary cells, but had no effect on non-mammary epithelial cells. These studies suggest that BRCA1 may normally serve as a negative regulator of mammary epithelial cell growth whose function is compromised in breast cancer either by direct mutation or alterations in gene expression.     

Increased sensitivity of gastric cancer cells to natural killer and lymphokine-activated killer cells by antisense suppression of N-acetylgalactosaminyltransferase

UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc transferases) catalyze the initial reaction in O-linked (mucin type) oligosaccharide biosynthesis. To attempt to inhibit the synthesis of O-glycan, we transfected antisense cDNA of GalNAc transferase type 1 (GalNAc-T1) into a human gastric cancer cell line, JRST. A decreased expression of GalNAc-T1 at the level of both mRNA and protein was observed in the resultant transfectants. They demonstrated a significantly increased sensitivity to NK and lymphokine-activated killer cells in vitro compared with parental cells and mock transfectants. Although there was no significant difference in in vitro cell proliferation among parental cells, mock transfectants, or antisense transfectants, the in vivo growth rate of antisense transfectants using SCID mice was clearly lower than that of parental cells and mock transfectants. Furthermore, the treatment of mice with anti-asialo-G(M1) Ab abolished this growth-inhibitory effect of antisense transfection. From these results, we conclude that antisense suppression of GalNAc-T1 could increase the sensitivity of tumor cells to NK and lymphokine-activated killer cells, suggesting that this strategy may be of use as a new immunogene therapy.     

Antisense insulin-like growth factor I transferred into a rat hepatoma cell line inhibits tumorigenesis by modulating major histocompatibility complex I cell surface expression

We have established a hepatocarcinoma cell line (LFCI2 A) that produces voluminous tumors when injected subcutaneously into syngeneic Commentry rats. These neoplastic cells express both insulin-like growth factors (IGF) I and II. When transfected with an episomal cassette-expressing IGF-I antisense RNA, the modified LFCI2 A cell lines become poorly tumorigenic and, when injected subcutaneously, are associated with inhibition of the growth of the parental tumoral cells and/or induction of regression of established tumors. By contrast, cell lines isolated after transfection with the IGF-II antisense-expressing vector were as tumorigenic as the parental cell lines. The results are discussed in terms of protective immunity induced by the tumoral cells transfected by the IGF-I antisense vector. In the transfected hepatocarcinoma cells that do not produce IGF-I, the expression of major histocompatibility complex class I antigen was increased at least 4-fold compared with parental cells. The introduction of these cells in vivo induced a tumor-specific immunity that was associated with CD8 T cells.     

c-Myc is a major mediator of the synergistic growth inhibitory effects of retinoic acid and interferon in breast cancer cells

The molecular signaling events involved in the inhibition of breast cancer cell growth by retinoic acid and interferon-alpha were investigated. All-trans-retinoic acid and interferon-alpha acted synergistically to inhibit growth of both the estrogen receptor-positive breast cancer cell line MCF-7 and the estrogen receptor-negative line BT-20. In MCF-7 cells, all-trans-retinoic acid potentiated the effects of interferon-alpha by up-regulating the expression of the RNA-dependent protein kinase (PKR). Consequently, the synergism between all-trans-retinoic acid and interferon-alpha down-regulated the expression of c-Myc, but not its functional partner, Max. Transfection of MCF-7 cells with a dominant-negative mutant of PKR relieved c-Myc down-regulation and cell growth inhibition, indicating that PKR is directly involved in c-Myc down-regulation and that c-Myc down-regulation is responsible for the inhibition of cell growth. Corresponding with c-Myc down-regulation, c-Myc.Max heterodimers bound to their consensus DNA sequence were undetectable in cells treated with all-trans-retinoic acid and interferon-alpha, indicating diminished c-Myc functionality. When c-Myc was overexpressed ectopically via a c-Myc expression vector, MCF-7 cells became resistant to growth inhibition by all-trans-retinoic acid plus interferon-alpha. These experiments define the following pathway as a major pathway in the synergistic growth inhibition of MCF-7 cells by all-trans-retinoic acid plus interferon-alpha: all-trans-retinoic acid + interferon-alpha --> upward arrow double-stranded RNA-dependent protein kinase --> downward arrow c-Myc --> cell growth inhibition.