BI-69A11 enhances susceptibility of colon cancer cells to mda-7/IL-24-induced growth inhibition by targeting Akt.

BACKGROUND: Akt and its downstream signalling pathways contribute to the aetiology and progression of colorectal carcinoma (CRC). Targeting the Akt pathway is an attractive strategy but few chemotherapeutic drugs have been used to treat CRC with only limited success. BI-69A11, a small molecule inhibitor of Akt, efficiently inhibits growth in melanoma cells. Melanoma differentiation associated gene-7 (mda-7)/interleukin-24 promotes cancer-selective apoptosis when delivered by a tropism-modified replication incompetent adenovirus (Ad.5/3-mda-7). However, Ad.5/3-mda-7 displays diminished antitumour efficacy in several CRC cell lines, which correlates with the expression of K-RAS. METHODS: The individual and combinatorial effect of BI-69A11 and Ad.5/3-mda-7 in vitro was studied by cell viability, cell cycle, apoptosis and invasion assays in HT29 and HCT116 cells containing wild type or mutant K-ras, respectively. In vivo HT29 tumour xenografts were used to test the efficacy of the combination treatment. RESULTS: BI-69A11 inhibited growth and induced apoptosis in CRC. However, combinatorial treatment was more effective compared with single treatment. This combination showed profound antitumour and anti angiogenic effects in vitro and in vivo by downregulating Akt activity. CONCLUSIONS: BI-69A11 enhances the antitumour efficacy of Ad.5/3-mda-7 on CRC overexpressing K-RAS by inducing apoptosis and regulating Akt activity thereby warranting further evaluation in treating CRC.

Reversibility of progression of the transformed phenotype in Ad5-transformed rat embryo cells.

The carcinogenic process is extremely complex and is affected by diverse environmental and host factors. The mechanism for the gradual development of the transformed phenotype (a process termed "progression") was studied in type 5 adenovirus (Ad5)-transformed rat embryo cells. Progression was not correlated with major changes in the pattern of integration of viral DNA sequences. Instead, it was associated with an increased methylation of integrated viral sequences other than those corresponding to the E1 transforming genes of Ad5. A single exposure of progressed cells to the demethylating agent 5-azacytidine (Aza) resulted in a stable reversion to the unprogressed state of the original parental clone. A further selection of cells after growth in agar allowed the isolation of Aza-treated clones that had regained the progressed phenotype. These observations indicate that progression is a reversible process and suggest that progression may be associated with changes in the state of methylation of one or more specific genes.

Recombinant interferon enhances monoclonal antibody-targeting of carcinoma lesions in vivo.

Heterogeneity in the expression of tumor-associated antigens, as defined by the binding of monoclonal antibodies, is a characteristic common to most, if not all, human carcinoma cell populations. Antigen-negative cells within the population can escape detection and therapy by their failure to bind the appropriate antibody. Therefore, the extent of antigenic heterogeneity is an important consideration when designing protocols for the management of cancer by administration of monoclonal antibodies. One approach to counteracting the effect of antigenic heterogeneity is the use of clone A of recombinant human leukocyte interferon (Hu-IFN-alpha A). Administration of Hu-IFN-alpha A in vivo effectively increased the amount of tumor antigen expressed by a human colon xenograft in situ and augmented the localization of a radiolabeled monoclonal antibody to the tumor site. Concomitant administration of Hu-IFN-alpha A and monoclonal antibody may thus be effective in overcoming the antigenic heterogeneity of carcinoma cell populations and in enhancing the efficacy of monoclonal antibodies in the detection and treatment of carcinoma lesions.

Targeting inhibition of K-ras enhances Ad.mda-7-induced growth suppression and apoptosis in mutant K-ras colorectal cancer cells.

Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) is a cancer-specific, growth-suppressing and apoptosis-inducing gene with broad-spectrum antitumor activity. However, when administered by means of a replication-incompetent adenovirus, Ad.mda-7, several colorectal carcinoma cell lines are resistant to its antiproliferative and antisurvival effects. We have presently endeavored to determine if K-ras mutations, present in approximately 40-50% of colorectal cancers and which may mediate resistance to chemotherapy and radiotherapy, represent a predisposing genetic factor mitigating reduced sensitivity to Ad.mda-7. To suppress ras expression, three structurally different replication-incompetent adenoviral vectors were engineered that express (1) an intracellular, neutralizing single-chain antibody (scAb) to p21 ras (Ad.K-ras scAb), (2) an antisense (AS) K-ras gene (Ad.K-ras AS) or (3) both mda-7/IL-24 and a K-ras AS gene in a single bipartite virus (Ad.m7.KAS). Simultaneous inhibition of K-ras and expression of mda-7/IL-24 enhanced killing of colorectal carcinoma cells with mutated K-ras, but not with wild-type K-ras. The extent of killing depended on the degree of K-ras downregulation, with Ad.K-ras AS being generally more efficient than Ad.K-ras scAb in combination with Ad.mda-7. These findings support an effective dual-combinatorial approach for the therapy of colorectal cancers that employs a unique cancer-specific suppressor gene (mda-7/IL-24) with targeted inhibition of oncogene (ras) expression.

Ionizing radiation enhances therapeutic activity of mda-7/IL-24: overcoming radiation- and mda-7/IL-24-resistance in prostate cancer cells overexpressing the antiapoptotic proteins bcl-xL or bcl-2.

Subtraction hybridization applied to terminally differentiating human melanoma cells identified mda-7/IL-24, a cytokine belonging to the IL-10 gene superfamily. Adenoviral-mediated delivery of mda-7/IL-24 (Ad.mda-7) provokes apoptosis selectively in a wide spectrum of cancers in vitro in cell culture, in vivo in human tumor xenograft animal models and in patients with advanced carcinomas and melanomas. In human prostate cancer cells, a role for mitochondrial dysfunction and induction of reactive oxygen species in the apoptotic process has been established. Ectopic overexpression of bcl-xL and bcl-2 prevents these changes including apoptosis induction in prostate tumor cells by Ad.mda-7. We now document that this resistance to apoptosis can be reversed by treating bcl-2 family overexpressing prostate tumor cells with ionizing radiation in combination with Ad.mda-7 or purified GST-MDA-7 protein. Additionally, radiation augments apoptosis induction by mda-7/IL-24 in parental and neomycin-resistant prostate tumor cells. Radiosensitization to mda-7/IL-24 is dependent on JNK signaling, as treatment with the JNK 1/2/3 inhibitor SP600125 abolishes this effect. Considering that elevated expression of bcl-xL and bcl-2 are frequent events in prostate cancer development and progression, the present studies support the use of ionizing radiation in combination with mda-7/IL-24 as a means of augmenting the therapeutic benefit of this gene in prostate cancer, particularly in the context of tumors displaying resistance to radiation therapy owing to bcl-2 family member overexpression.

Defining the mechanism by which IFN-beta dowregulates c-myc expression in human melanoma cells: pivotal role for human polynucleotide phosphorylase (hPNPaseold-35).

Type I interferons (IFN-alpha/-beta) are capable of suppressing c-myc mRNA expression by modulating post-transcriptional processing. However, the molecular mechanism of this phenomenon is poorly understood. We previously established that human polynucleotide phosphorylase (hPNPase(old-35)), a type I IFN-inducible 3',5' exoribonuclease involved in mRNA degradation, induces G1 cell cycle arrest and eventually apoptosis by specifically degrading c-myc mRNA. We now demonstrate a close association between IFN-beta-induced hPNPase(old-35) upregulation and c-myc downregulation in human melanoma cells. Employing stable melanoma cell clones expressing hPNPase(old-35) small inhibitory RNA, we demonstrate that hPNPase(old-35) is a key molecule coupled with IFN-beta-mediated downregulation of c-myc mRNA. Inhibition of hPNPase(old-35) or overexpression of c-myc protects melanoma cells from IFN-beta-mediated growth inhibition, emphasizing the importance of hPNPase(old-35) upregulation and consequent c-myc downregulation in IFN-beta-induced growth inhibition and apoptosis induction. In these contexts, targeted overexpression of hPNPase(old-35) might be a novel therapeutic strategy for c-myc-overexpressing and IFN-resistant tumors, such as melanomas.

Activation of Ras/Raf protects cells from melanoma differentiation-associated gene-5-induced apoptosis.

Melanoma differentiation-associated gene-5 (mda-5) was the first molecule identified in nature whose encoded protein embodied the unique structural combination of an N-terminal caspase recruitment domain and a C-terminal DExD/H RNA helicase domain. As suggested by its structure, cumulative evidences documented that ectopic expression of mda-5 leads to growth inhibition and/or apoptosis in various cell lines. However, the signaling pathways involved in mda-5-mediated killing have not been elucidated. In this study, we utilized either genetically modified cloned rat embryo fibroblast cells overexpressing different functionally and structurally distinct oncogenes or human pancreatic and colorectal carcinoma cells containing mutant active ras to resolve the role of the Ras/Raf signaling pathway in mda-5-mediated growth inhibition/apoptosis induction. Rodent and human tumor cells containing constitutively activated Raf/Raf/MEK/ERK pathways were resistant to mda-5-induced killing and this protection was antagonized by intervening in this signal transduction cascade either by directly inhibiting ras activity using an antisense strategy or by targeting ras-downstream factors, such as MEK1/2, with the pharmacological inhibitor PD98059. The present findings provide a further example of potential cross-talk between growth-inhibitory and growth-promoting pathways in which the ultimate balance of these factors defines cellular homeostasis, leading to survival or induction of programmed cell death.

Analysis of the reduced growth factor dependency of simian virus 40-transformed 3T3 cells.

We have measured in a defined serum-free medium the platelet-derived growth factor (PDGF) and insulin requirements of normal Swiss 3T3 cells, simian virus 40-transformed 3T3 cells, and partial revertants of simian virus 40-transformed 3T3 cells. Swiss 3T3 cells displayed strong requirements for both PDGF and insulin. Both of these requirements were significantly diminished in simian virus 40-transformed 3T3 cells. Analysis of the PDGF and insulin requirements of the revertants indicated that the loss of either of these two growth factor requirements was not necessarily linked to the other; rather, the growth factor requirements were specifically associated with other parameters of transformation. The reacquisition of a PDGF requirement cosegregated with reversion to density-dependent growth inhibition, whereas reacquisition of a normal insulin requirement cosegregated with reversion to a normal growth dependence on calf serum. Anchorage dependence was dissociable from both growth factor requirements. The relationship between the PDGF requirement and density-dependent growth inhibition was further analyzed in normal 3T3 cells by measuring the PDGF requirement at different cell densities. At high cell densities, the requirement for PDGF became significantly greater. We suggest that at least in part the ability of transformed cells to grow to high saturation densities results from their loss of a requirement for PDGF.

Accelerated onset of viral transcription in adenovirus-infected HeLa cells treated with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate.

When adenovirus type 5-infected HeLa cells were exposed to the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate, short pulse-labeling with [3H]uridine in vivo and [3H]UTP incorporation by isolated nuclei in vitro were both consistent with a decreased latent period before initiation by RNA polymerase at early viral promoters. Acceleration was not dependent upon concurrent protein synthesis and could not be attributed to rapid entry of virus into the cell nucleus. 12-O-tetradecanoyl-phorbol-13-acetate suppressed the transcription-delay phenotype of the E1a mutant, hr1, without restoring its ability to replicate.

Tumor promoters alter the temporal program of adenovirus replication in human cells.

In this study we evaluated the effect of phorbol ester tumor promoters on the kinetics of adenovirus type 5 (Ad5) replication in human cells. When added at the time of infection, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) accelerated the appearance of an early virus antigen (72,000-molecular-weight [72K] deoxyribonucleic acid-binding protein), the onset of viral deoxyribonucleic acid synthesis, and the production of infectious virus. The appearance of an Ad5-specific cytopathic effect (CPE) was also accelerated in infected cultures exposed to TPA, whereas phorbol, 4 alpha-phorbol-12,13-didecanoate and 4-OmeTPA, which are inactive as tumor promoters, were ineffective in inducing this morphological change. The acceleration of the CPE seen in TPA-treated Ad5-infected cells was not caused by TPA induction of the protease plasminogen activator, since the protease inhibitors leupeptin and antipain do not inhibit the earlier onset of this CPE and, in contrast, epidermal growth factor, which induces plasminogen activator in HeLa cells, does not induce an earlier CPE. Evidence for a direct effect of TPA on viral gene expression was obtained by analyzing viral messenger ribonucleic acid (mRNA) synthesis. TPA accelerated the appearance of mRNA from all major early regions of Ad5, transiently stimulated the accumulation of region III mRNA, and accelerated the appearance of late Ad5 mRNA. Thus, TPA altered the temporal program of Ad5 mRNA production and accelerated the appearance of at least some Ad5-specific polypeptides during lytic infection of human cells. These effects presumably explain the earlier onset of the Ad5-specific CPE in TPA-treated cells and may have relevance to the effects of TPA on viral gene expression in nonpermissive cells carrying integrated viral deoxyribonucleic acid sequences.

Suppression of the progression phenotype in somatic cell hybrids occurs in the absence of altered adenovirus type 5 gene expression.

In the present study we have analyzed the genetic regulation of increased expression of transformation-associated traits, a process termed progression, in adenovirus type 5 (Ad5)-transformed secondary rat embryo cells. Somatic cell hybrids were formed between a highly progressed neomycin-resistant Ad5-transformed cloned cell line (E11-NMTneo) and an untransformed chloramphenicol-resistant rat embryo fibroblast cell line (CREFcap). Parental E11-NMTneo cells grew with high efficiency in agar, exhibited reduced 125I-epidermal growth factor (EGF) binding, and were tumorigenic in nude mice. Parental CREFcap cells exhibited phenotypes opposite to those of E11-NMTneo cells. A high proportion (84%) of the presumptive hybrid cell types obtained after fusion and genetic selection (G418 and chloramphenicol) displayed a flat morphological phenotype intermediate between CREFcap and E11-NMTneo cells, suggesting that a trans-dominant extinction phenomenon had occurred. Two hybrids with a round morphology (R), which still exhibited the progressed phenotype, and two hybrids with a flat morphology (F), which had lost the progressed phenotype, were chosen for detailed analysis. Both R hybrids grew efficiently in agar, exhibited low 125I-EGF binding, and were tumorigenic in nude mice, whereas both F hybrids grew poorly in agar, displayed increased 125I-EGF binding in comparison with E11-NMTneo and R hybrids, and were nontumorigenic in nude mice. An analysis of the viral DNA integration patterns and the rates of transcription, steady-state mRNA accumulation, and relative levels of the Ad5 E1A and E1B gene products revealed no differences among the parental and hybrid cells. These studies indicate that normal CREF cells may contain a suppressor gene(s) which can inhibit the expression of specific traits of the progression phenotype in Ad5-transformed cells and that this suppression is not associated with changes in the expression of Ad5 transforming genes.

Roles for basal and stimulated p21(Cip-1/WAF1/MDA6) expression and mitogen-activated protein kinase signaling in radiation-induced cell cycle checkpoint control in carcinoma cells.

We investigated the role of the cdk inhibitor protein p21(Cip-1/WAF1/MDA6) (p21) in the ability of MAPK pathway inhibition to enhance radiation-induced apoptosis in A431 squamous carcinoma cells. In carcinoma cells, ionizing radiation (2 Gy) caused both primary (0-10 min) and secondary (90-240 min) activations of the MAPK pathway. Radiation induced p21 protein expression in A431 cells within 6 h via secondary activation of the MAPK pathway. Within 6 h, radiation weakly enhanced the proportion of cells in G(1) that were p21 and MAPK dependent, whereas the elevation of cells present in G(2)/M at this time was independent of either p21 expression or MAPK inhibition. Inhibition of the MAPK pathway increased the proportion of irradiated cells in G(2)/M phase 24-48 h after irradiation and enhanced radiation-induced apoptosis. This correlated with elevated Cdc2 tyrosine 15 phosphorylation, decreased Cdc2 activity, and decreased Cdc25C protein levels. Caffeine treatment or removal of MEK1/2 inhibitors from cells 6 h after irradiation reduced the proportion of cells present in G(2)/M phase at 24 h and abolished the ability of MAPK inhibition to potentiate radiation-induced apoptosis. These data argue that MAPK signaling plays an important role in the progression/release of cells through G(2)/M phase after radiation exposure and that an impairment of this progression/release enhances radiation-induced apoptosis. Surprisingly, the ability of irradiation/MAPK inhibition to increase the proportion of cells in G(2)/M at 24 h was found to be dependent on basal p21 expression. Transient inhibition of basal p21 expression increased the control level of apoptosis as well as the abilities of both radiation and MEK1/2 inhibitors to cause apoptosis. In addition, loss of basal p21 expression significantly reduced the capacity of MAPK inhibition to potentiate radiation-induced apoptosis. Collectively, our data argue that MAPK signaling and p21 can regulate cell cycle checkpoint control in carcinoma cells at the G(1)/S transition shortly after exposure to radiation. In contrast, inhibition of MAPK increases the proportion of irradiated cells in G(2)/M, and basal expression of p21 is required to maintain this effect. Our data suggest that basal and radiation-stimulated p21 may play different roles in regulating cell cycle progression that affect cell survival after radiation exposure.

A role for both Ets and C/EBP transcription factors and mRNA stabilization in the MAPK-dependent increase in p21 (Cip-1/WAF1/mda6) protein levels in primary hepatocytes.

In primary hepatocytes and HepG2 hepatoma cells, prolonged activation of the p42/44 mitogen-activated protein kinase (MAPK) pathway is associated with a reduction in DNA synthesis, mediated by increased expression of the cyclin-dependent kinase inhibitor protein p21 (Cip-1/WAF1/mda6) (p21). This study was performed to evaluate the contribution of transcriptional and post-transcriptional regulation in this response. Prolonged activation of the MAPK pathway in wild-type or p21 null hepatocytes caused a large decrease and increase, respectively, in DNA synthesis. Prolonged activation of the MAPK pathway in either wild-type or p21 antisense HepG2 cells also caused large decreases and increases, respectively, in DNA synthesis. MAPK signaling increased the phosphorylation of the transcription factors Ets2, C/EBPalpha, and C/EBPbeta, and rapidly increased transcription from the p21 promoter via multiple Ets- and C/EBP-elements within the enhancer region. Eight hours after MAPK activation, loss of C/EBPbeta or Ets2 function significantly reduced MAPK-stimulated transcription from the p21 promoter and abolished increased p21 protein expression. At this time, MAPK signaling increased both p21 mRNA and p21 protein stabilities that were also demonstrated to be essential for a profound increase in p21 protein levels. Thirty-six hours after MAPK activation, transcription from the p21 promoter was still significantly reduced in cells without either C/EBPbeta or Ets2 function; however, these cells were now capable of exhibiting a partial increase in p21 protein expression. In contrast, loss of C/EBPalpha function modestly reduced MAPK-stimulated transcription from the p21 promoter but strongly inhibited the ability of prolonged MAPK activation to increase protein levels of p21. This data suggested that prolonged enhancement of p21 protein levels may be under posttranscriptional control. In agreement with this hypothesis, prolonged MAPK signaling further increased p21 mRNA stability at 36 h, compared with the 8-h time point. Our data argue that MAPK signaling increased p21 promoter activity via multiple transcription factors, which alone were insufficient for a robust prolonged increase in p21 protein levels in primary hepatocytes, and that to increase p21 protein levels also required enhanced stabilization of p21 mRNA and p21 protein. Collectively, these data suggest that loss of transcription factor and mRNA/protein stabilization functions correlates with an inability of MAPK signaling to cause growth arrest versus proliferation in primary hepatocytes.

Deoxycholic acid (DCA) causes ligand-independent activation of epidermal growth factor receptor (EGFR) and FAS receptor in primary hepatocytes: inhibition of EGFR/mitogen-activated protein kinase-signaling module enhances DCA-induced apoptosis.

Previous studies have argued that enhanced activity of the epidermal growth factor receptor (EGFR) and the mitogen-activated protein kinase (MAPK) pathway can promote tumor cell survival in response to cytotoxic insults. In this study, we examined the impact of MAPK signaling on the survival of primary hepatocytes exposed to low concentrations of deoxycholic acid (DCA, 50 microM). Treatment of hepatocytes with DCA caused MAPK activation, which was dependent upon ligand independent activation of EGFR, and downstream signaling through Ras and PI(3) kinase. Neither inhibition of MAPK signaling alone by MEK1/2 inhibitors, nor exposure to DCA alone, enhanced basal hepatocyte apoptosis, whereas inhibition of DCA-induced MAPK activation caused approximately 25% apoptosis within 6 h. Similar data were also obtained when either dominant negative EGFR-CD533 or dominant negative Ras N17 were used to block MAPK activation. DCA-induced apoptosis correlated with sequential cleavage of procaspase 8, BID, procaspase 9, and procaspase 3. Inhibition of MAPK potentiated bile acid-induced apoptosis in hepatocytes with mutant FAS-ligand, but did not enhance in hepatocytes that were null for FAS receptor expression. These data argues that DCA is causing ligand independent activation of the FAS receptor to stimulate an apoptotic response, which is counteracted by enhanced ligand-independent EGFR/MAPK signaling. In agreement with FAS-mediated cell killing, inhibition of caspase function with the use of dominant negative Fas-associated protein with death domain, a caspase 8 inhibitor (Ile-Glu-Thr-Asp-p-nitroanilide [IETD]) or dominant negative procaspase 8 blocked the potentiation of bile acid-induced apoptosis. Inhibition of bile acid-induced MAPK signaling enhanced the cleavage of BID and release of cytochrome c from mitochondria, which were all blocked by IETD. Despite activation of caspase 8, expression of dominant negative procaspase 9 blocked procaspase 3 cleavage and the potentiation of DCA-induced apoptosis. Treatment of hepatocytes with DCA transiently increased expression of the caspase 8 inhibitor proteins c-FLIP-(S) and c-FLIP-(L) that were reduced by inhibition of MAPK or PI(3) kinase. Constitutive overexpression of c-FLIP-(s) abolished the potentiation of bile acid-induced apoptosis. Collectively, our data argue that loss of DCA-induced EGFR/Ras/MAPK pathway function potentiates DCA-stimulated FAS-induced hepatocyte cell death via a reduction in the expression of c-FLIP isoforms.

PEA3 sites within the progression elevated gene-3 (PEG-3) promoter and mitogen-activated protein kinase contribute to differential PEG-3 expression in Ha-ras and v-raf oncogene transformed rat embryo cells.

Transformation of normal cloned rat embryo fibroblast (CREF) cells with cellular oncogenes results in acquisition of anchorage-independent growth and oncogenic potential in nude mice. These cellular changes correlate with an induction in the expression of a cancer progression-promoting gene, progression elevated gene-3 (PEG-3). To define the mechanism of activation of PEG-3 as a function of transformation by the Ha-ras and v-raf oncogenes, evaluations of the signaling and transcriptional regulation of the approximately 2.0 kb promoter region of the PEG-3 gene, PEG-Prom, was undertaken. The full-length and various mutated regions of the PEG-Prom were linked to a luciferase reporter construct and tested for promoter activity in CREF and oncogene-transformed CREF cells. An analysis was also performed using CREF cells doubly transformed with Ha-ras and the Ha-ras specific suppressor gene Krev-1, which inhibits the transformed phenotype in vitro. These assays document an association between expression of the transcription regulator PEA3 and PEG-3. The levels of PEA3 and PEG-3 RNA and proteins are elevated in the oncogenically transformed CREF cells, and reduced in transformation and tumorigenic suppressed Ha-ras/Krev-1 doubly transformed CREF cells. Enhanced tumorigenic behavior, PEG-3 promoter function and PEG-3 expression in Ha-ras transformed cells were all dependent upon increased activity within the mitogen-activated protein kinase (MAPK) pathway. Electrophoretic mobility shift assays and DNase I footprinting experiments indicate that PEA3 binds to sites within the PEG-Prom in transformed rodent cells in an area adjacent to the TATA box in a MAPK-dependent fashion. These findings demonstrate an association between Ha-ras and v-raf transformation of CREF cells with elevated PEA3 and PEG-3 expression, and they implicate MAPK signaling via PEA3 as a signaling cascade involved in activation of the PEG-Prom.

Use of the human EF-1alpha promoter for expression can significantly increase success in establishing stable cell lines with consistent expression: a study using the tetracycline-inducible system in human cancer cells.

Establishing cells with an exogenously introduced gene of interest under the inducible control of tetracycline (Tc) initially requires clonal cell lines stably expressing the tetracycline activator (tTA or rtTA). The originally described plasmid vectors expressing tTA/rtTA are driven by the cytomegalovirus (CMV) immediate early (IE) promoter-enhancer, known for its robust activity in a wide spectrum of cell types. While many reports testify to the utility and efficacy of this construct, instances of inexplicable failure to establish cell lines having inducible expression of the cDNA under study are encountered. Spontaneous extinction of CMV promoter activity in cells has been observed in a temporal and cell type-dependent manner. This could be a contributing factor in the failure to establish Tc-responsive cell lines. We here report that a change of the expression cassette to the human elongation factor-1alpha (EF-1alpha) promoter has permitted successful establishment of several inducible cell lines from diverse human tumor tissue origins. We interpret these results to imply that extinction of rtTA (or tTA) expression might be a significant factor in the lack of success in establishing Tc-inducible cell lines. Moreover, the present findings have general relevance to experiments requiring the use of stable cell lines.

Surface properties of normal, transformed, and temperature-sensitive rat liver epithelial cells.

Cell surface proteins, glycoproteins, and glycopeptides from normal, transformed, and mutant temperature-sensitive rat liver epithelial cells were radiolabeled and compared by Sephadex G-50 chromatography and polyacrylamide gel electrophoresis. Some changes were observed in the transformed cell lines including an increase in high-molecular-weight glycopeptides and a decrease in the amount of fibronectin (large external transformation-sensitive) protein. The mutant TS223 cells, which were temperature-sensitive for maintenance of the transformed phenotype when grown at the nonpermissive temperature (41 degrees C), had characteristics intermediate between those of the normal and transformed phenotypes. These results corresponded to those of previous studies indicating that TS223 was somewhat "leaky" at 41 degrees C; i.e., although more "normal" than at 37 degrees C, it was not completely normal. Alterations in surface macromolecules in transformed epithelial cells may, therefore, represent a useful marker for transformation because morphologic changes are usually not readily apparent in transformed epithelial cells.

Surface-epitope masking: a strategy for the development of monoclonal antibodies specific for molecules expressed on the cell surface.

BACKGROUND: Producing monoclonal antibodies against specific targets, including tumor-specific antigens, is a tedious and extremely inefficient process. PURPOSE: Our purpose was to determine whether DNA transfection combined with an immunologic masking tactic could be used to efficiently generate hybridomas that secrete monoclonal antibodies. The quest was for monoclonal antibodies that would react with molecules existing on the surface of genetically altered cells. METHODS: We developed a masking technique called surface-epitope masking (SEM). The SEM procedure involves the selective blocking of surface antigens present in a genetically engineered cell (referred to as a "tester") with high-titer polyclonal antibodies that have been produced against the untransfected parental cell (referred to as a "driver"). Surface-epitope-masked tester cells were injected into BALB/c mice; immune spleen cells then taken from these mice were fused with myeloma cells. RESULTS: This process resulted in the efficient generation of hybridomas that secreted monoclonal antibodies that reacted with cell-surface antigens on transfected tester cells and with additional cell types that expressed the same surface molecules. In one case, CREF-Trans 6 cells were engineered to express a typical multidrug-resistant (MDR) phenotype. Using CREF-Trans 6:MDR cells as a tester cell line, we utilized the SEM procedure to produce monoclonal antibodies that displayed surface reactivity to both CREF-Trans 6:MDR cells and MDR human breast carcinoma (MCF7) cells. In a second case, human prostatic carcinoma CREF-Trans 6 cells, which were DNA transfected and derived from nude mouse tumors, were used as the tester cell line. The SEM procedure was again used to produce monoclonal antibodies. These antibodies were designed to and did react with: (a) tumor-associated antigens on the surface of the original LNCaP cell line used to obtain human prostatic carcinoma DNA, (b) primary and secondary nude mouse transfectants derived from tumors, and (c) two additional human prostatic carcinoma cell lines, DU-145 and PC-3. CONCLUSIONS: The SEM approach was used for the efficient and selective development of monoclonal antibodies that react with cell-surface molecules with both known and unknown functions. IMPLICATIONS: The SEM procedure should be useful in producing monoclonal antibodies and identifying genes associated with important cellular processes, including immunologic recognition, tumorigenesis, metastasis, atypical multidrug resistance, and autoimmune diseases.

Effect of recombinant fibroblast interferon and recombinant immune interferon on growth and the antigenic phenotype of multidrug-resistant human glioblastoma multiforme cells.

To study the effect of drug resistance on the response of stage IV astrocytomas to interferon, a human glioblastoma multiforme cell line, GBM-18, was transfected with an expression-vector plasmid containing a human multidrug resistance (MDR) gene (pHaMDR1/A), and clones surviving in colchicine were isolated. GBM-18 multidrug-resistant subclones displayed cross-resistance to other chemotherapeutic agents, including vincristine, doxorubicin, and dactinomycin. The multidrug-resistant phenotype was reversible when GBM-18 multidrug-resistant cells were cultured in colchicine and the calcium-channel blocker verapamil. The level of the MDR1 gene (also known as PGY1) message was increased in GBM-18 multidrug-resistant cells selected for increased resistance to colchicine, and this effect was not correlated with an amplification of the MDR1 gene. In both parental GBM-18 and GBM-18 multidrug-resistant cells, growth was suppressed to a greater degree when cultures were treated with the combination of fibroblast interferon (IFN-beta) and immune interferon (IFN-gamma). Parental cells and multidrug-resistant subclones varied in their de novo and/or interferon-modulated expression of HLA class I and class II antigens, a high-molecular-weight melanoma-associated antigen, and intercellular adhesion molecule 1 (ICAM-1). Of the antigens tested, ICAM-1 and HLA class I antigens were the most sensitive to enhanced expression induced by IFN-beta and IFN-gamma when used alone or in combination. The results of the present study indicate that multidrug-resistant human glioblastoma multiforme cells retain their increased sensitivity to the antiproliferative activity of the combination of IFN-beta plus IFN-gamma, and differences in antigenic phenotype are apparent in independent multidrug-resistant glioblastoma multiforme clones.

Effect of phorbol ester tumor promoters on the expression of melanogenesis in B-16 melanoma cells.

Cells of the C3 clone of B-16 melanoma synthesize melanin only at confluence after which they senesce and can no longer be passaged. Addition to the cultures of 10(-8)--10(-7) M 12-O-tetradecanoylphorbol-13-acetate (TPA) shortly after plating delayed by about 2 days the onset of melanogenesis. TPA did not, however, affect the growth of the cells or the time at which they reached confluence. The ability of a series of phorbol esters to delay melanogenesis correlated with their tumor-promoting activity on mouse skin. The optimum time for addition of TPA was within the first 24 hr after plating; the inhibitory effect decreased when TPA was added at later points. alpha-melanocyte-stimulating hormone (5 x 10(-7) M) added to B-16 cultures 24 hr after plating slowed the growth of the cells and caused them to differentiate when still subconfluent. TPA also inhibited this alpha-melanocyte-stimulating hormone-induced melanogenesis. These results suggest that TPA inhibits a very early stage in a stepwise process that leads to the differentiation of these cultures. For reasons that are not apparent, the cells eventually escape from this inhibition. The B-16 melanoma cell culture system may be useful for studying the mechanism by which TPA and related tumor promoters affect cellular differentiation.