Inhibition of CRIPTO expression and tumorigenicity in human colon cancer cells by antisense RNA and oligodeoxynucleotides.

CRIPTO is an epidermal growth factor-related gene expressed in a majority of human colorectal tumors. To assess the role of CRIPTO in the growth control of human colon cancer, we have treated human colon carcinoma GEO and CBS cells, that possess high levels of CRIPTO, and WIDR colon cancer cells, that are negative for CRIPTO expression, with two antisense phosphorothioate oligodeoxynucleotides complementary to the 5' end of the human CRIPTO mRNA. Both antisense oligodeoxynucleotides significantly reduced endogenous CRIPTO protein levels and inhibited GEO and CBS cell growth in monolayer and in semisolid medium, whereas they did not affect WIDR cell growth. In addition, GEO, CBS and WIDR cells were infected with a recombinant retroviral vector containing the hygromycin-resistance gene and a 900 bp EcoRI-EcoRI coding fragment of the human CRIPTO cDNA oriented in the 3' to 5' direction. GEO and CBS CRIPTO antisense infectants exhibited a 60 to 70% reduction in CRIPTO protein expression, in monolayer growth and in soft agar cloning efficiency as compared to parental noninfected cells. In contrast, infection of WIDR cells with the CRIPTO antisense retrovirus did not alter their growth. Finally, GEO CRIPTO antisense infectants formed tumors in nude mice that were significantly smaller and had a larger latency period as compared to noninfected GEO cells.

Growth inhibition of human colon carcinoma cells by combinations of anti-epidermal growth factor-related growth factor antisense oligonucleotides.

GEO is a well-differentiated colon cancer cell line that coexpresses the epidermal growth factor-like growth factors CRIPTO (CR), amphiregulin (AR), and transforming growth factor alpha (TGF-alpha). Antisense 20-mer phosphorothioate oligodeoxynucleotides (AS S-oligos) directed against CR, AR, and TGF-alpha mRNAs were equipotent in their ability to inhibit both the anchorage-dependent growth and the anchorage-independent growth (AIG) of GEO cells, with a 50% inhibitory concentration of about 5 micrometer in the AIG assay. A supraadditive effect was observed when a combination of S-oligos was used. For example, a combination of two different AS S-oligos (either AR + CR, or TGF-alpha + CR, or TGF-alpha + AR) at a concentration of 1 micrometer each (total concentration, 2 micrometer) resulted in 50% inhibition of GEO cells AIG, whereas the use of each AS S-Oligo at a 1 or 2 micrometer concentration resulted respectively in about 10 and 20% growth inhibition. A combination of the three AS S-oligos was even more effective, resulting in about 60% inhibition of GEO cells AIG at a concentration of 1 micrometer each (3 micrometer total concentration). The AS S-oligos were also able to inhibit specifically the expression of either AR, CR, or TGF-alpha proteins in GEO cells, as assessed using immunocytochemistry or Western blot analysis. Finally, a supraadditive growth inhibitory effect of the AS S-oligos and an epidermal growth factor receptor-blocking antibody (monoclonal antibody 528) was observed. These data suggest that the use of a combination of AS S-oligos directed against different growth factors and antibodies directed against their receptors might result in an efficient inhibition of colon carcinoma cell growth.

Inhibition of HIV-1 replication in H9 cells by nystatin-A compared with other antiviral agents.

Nystatin A was compared in vitro with amphotericin B, AZT, or foscarnet for their respective abilities to inhibit the replication of human immunodeficiency virus type 1 (HIV-1) in H9 cells. HIV-1-infected H9 cells were cultured for 7 days in the presence of each of these drugs, at various concentrations. Reverse transcriptase activity and p24 antigen production were quantitated. Untreated, HIV-1-infected H9 cells served as the control. Nystatin A inhibited viral replication most effectively at 10 micrograms/ml, a concentration that did not affect cell viability. Nystatin-A treatment inhibited RT activity by 85% and p24 production by 90%. These levels of inhibition were comparable to that mediated by amphotericin B, AZT, or foscarnet at 10, 25, and 50 micrograms/ml, respectively. Western blot analysis of the HIV-1-infected H9 cells treated with these drugs did not detect any expression of viral proteins. These findings were further corroborated by indirect immunofluorescence studies using monoclonal anti-gp120 FITC-conjugated antibodies and by polymerase chain reaction for proviral DNA analysis, using a 32P-labeled probe. These results suggest that Nystatin A merits attention as an antiviral drug for the treatment of HIV-1 infection. In vivo drug delivery by liposome encapsulation to overcome problems of bioavailability is currently under study.

Inhibition of HIV replication by immunoliposomal antisense oligonucleotide.

The sequence-specific suppression of HIV-1 replication using CD4 monoclonal-antibody-targeted liposomes, containing Rev antisense phosphorothioate oligonucleotides is described. Liposomes were prepared by encapsulating the 20-mer antisense DNA sequence of the rev HIV-1 regulatory gene, in the form of a phosphorothioate oligonucleotide. Specific targeting was accomplished by conjugating anti-CD4 mouse monoclonal antibody to the surface of the liposomes. HIV-1-infected H9 cells as well as peripheral blood T-lymphocytes were incubated with the immunoliposomes of antisense found to have potential antiviral effect. HIV-1 replication was reduced by 85% in antisense immunoliposome-treated H9 cells and peripheral blood lymphocytes, whereas the inhibition of HIV-1 replication was not observed using either empty immunoliposomes or immunoliposomes containing scrambled Rev phosphorothioate oligonucleotide sequences. The antiviral activity of both the free and the encapsulated oligonucleotides were assessed by p24, reverse transcriptase (RT) assays and polymerase chain reaction (PCR) analysis. Liposome preparations demonstrated minimal toxicity in H9 as well as in peripheral blood lymphocyte cell culture experiments. These in vitro culture results demonstrate the potential efficacy of immunoliposomes to inhibit HIV replication.

A novel, sensitive radioimmunoprecipitation assay for the detection of antibodies to human immunodeficiency virus-type 2.

A radioimmunoprecipitation assay that provides an alternative to the Western blot assay was developed for characterizing antibodies against human immunodeficiency virus type-2 (HIV-2). The assay is based on radioiodination of antigen using Bolton-Hunter reagent. The antigen consists of a soluble preparation of the NIH-Z (HIV-2) strain of 1000X purified virus spiked with purified recombinant HIV-2 gp105. Radiolabeled proteins were immunoprecipitated by immune human sera, even at the early stages of seroconversion. This new assay provides a simple method for characterizing and titrating antibodies against HIV-2. The method is more sensitive, and is more efficient than Western blotting. The labeled viral proteins are well suited for biochemical studies.

Down-regulation of RI alpha subunit of cAMP-dependent protein kinase induces growth inhibition of human mammary epithelial cells transformed by c-Ha-ras and c-erbB-2 proto-oncogenes.

MCF-10A is a spontaneously immortalized, non-transformed human mammary epithelial cell line. We have recently obtained MCF-10A clones (MCF-10A HE cells) that are transformed following over-expression of both a human point-mutated c-Ha-ras and the c-erbB-2 proto-oncogenes. Two isoforms of the cAMP-dependent protein kinase (cAK) have been described in mammalian cells. Enhanced levels of type-I cAK (cAKI) are generally found in tumor cells. To determine whether inhibition of cAKI expression may interfere with ras and erbB-2 oncogene-induced transformation of human mammary epithelial cells, we have tested the effects of 2 agents that specifically down-regulate cAKI, such as 8-chloro-cAMP and an anti-sense oligodeoxynucleotide targeted against the RI alpha regulatory subunit of cAKI on MCF-10A HE cells. Treatment of MCF-10A HE cells with 8-chloro-cAMP induces a dose-dependent growth inhibition under both monolayer and soft-agar growth conditions, that is correlated with an accumulation of MCF-10A HE cells in G0/G1 phases of the cell cycle and a reduction of the number of cells in S phase. In contrast, 8-chloro-cAMP has no effect on MCF-10A cell growth. Furthermore, 8-chloro-cAMP treatment of MCF-10A HE cells induces a 4- to 6-fold reduction in p185erbB-2 expression and brings p21ras expression to levels comparable to those found in MCF-10A cells. Treatment of MCF-10A HE cells with an RI alpha anti-sense oligodeoxynucleotide determines a comparable inhibition of both anchorage-dependent and anchorage-independent cell growth. Our results suggest that cAKI may act as a mediator of ras and erbB-2 oncogene action in human breast cells and that interference with cAKI action provides a potential tool for inhibiting the growth-promoting effects of these oncogenes.

Amphiregulin as an autocrine growth factor for c-Ha-ras- and c-erbB-2-transformed human mammary epithelial cells.

Amphiregulin (AR), a member of the epidermal growth factor (EGF) family, was found to be as potent as EGF in stimulating the anchorage-dependent growth (ADG) of immortalized, nontransformed human mammary epithelial MCF-10A cells. MCF-10A cells transformed by either an activated human c-Ha-ras protooncogene (MCF-10A ras) or by overexpression of a nonactivated rat c-neu gene (MCF-10A neu) exhibited a 35% reduction in the response to AR in ADG when compared to MCF-10A cells, but AR was still as potent as EGF in these transformants. Exogenous AR exhibited only 15-20% of the activity of EGF in stimulating the anchorage-independent growth, a response that is normally dependent upon exogenous EGF, of the oncogene-transformed MCF-10A cells. MCF-10A cells express low levels of a 1.4-kb AR mRNA transcript, while MCF-10A ras and MCF-10A neu cells display a 15- to 30-fold increase in the levels of AR mRNA and endogenous AR protein as determined by Western blot analysis. Exogenous EGF was found to induced both the AR mRNA and protein in the MCF-10A parental and transformed cells. A 20-mer phosphorothioate antisense deoxyoligonucleotide complementary to the 5' sequence of AR mRNA was able to significantly reduce the levels of endogenous AR protein and to inhibit the EGF-stimulated ADG and anchorage-independent growth of MCF-10A ras and MCF-10A neu cells. These data suggest that AR may function as an EGF-dependent autocrine growth factor in mammary epithelial cells that have been transformed by either a point-mutated c-Ha-ras or c-neu.

Anti-sense oligonucleotides directed against EGF-related growth factors enhance anti-proliferative effect of conventional anti-tumor drugs in human colon-cancer cells.

We have demonstrated that anti-sense phosphorothioate oligodeoxynucleotides (AS S-oligos) directed against the EGF-like growth factors CRIPTO (CR), amphiregulin (AR) or transforming-growth-factor-alpha(TGFalpha) mRNA, are equipotent in their ability to inhibit the growth of human colon-carcinoma GEO cells. In this study, we evaluated the effect of combinations of these AS S-oligos and conventional anti tumor drugs, such as 5-fluorouracil (5-FU), adriamycin (ADR), mitomycin C (MIT) and cis-platinum (CDDP), on GEO cell growth. Dose-dependent growth inhibition was observed by treatment either with AS S-oligos or with anti-tumor drugs, using a clonogenic assay. Furthermore, an additive growth inhibitory effect occurred when GEO cells were exposed to the AS S-oligos after treatment with different concentrations of either 5-FU, MIT, ADR or CDDP. For example, treatment of GEO cells with a combination of low concentrations of 5-FU and any of the 3 AS S-oligos resulted in up to 70% growth inhibition. However, treatment of GEO cells with AS S-oligos before exposure to 5-FU or CDDP resulted in reduced efficacy of both drugs. Flow-cytometric analysis of DNA content demonstrated that treatment with the AS S-oligos caused a slight reduction of the percentage of cells in the S-phase of the cell cycle. These data suggest that combinations of AS S-oligos directed against EGF-related growth factors and of conventional anti-tumor drugs may result in efficient inhibition of colon-carcinoma cell growth.

Amphiregulin anti-sense oligodeoxynucleotides inhibit growth and transformation of a human colon carcinoma cell line.

Amphiregulin (AR) is a secreted heparin-binding growth factor that is structurally and functionally related to epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha). GEO cells are from a human colon cancer cell line that expresses high levels of AR protein and mRNA. To assess the role of AR in colon-cancer cell proliferation and transformation, 2 different anti-sense 20-mer phosphorothioate oligodeoxynucleotides (AR AS-1 and AR AS-2 S-oligos) complementary to the 5' sequence of AR mRNA were synthesized. Both AR AS S-oligos were able to inhibit the anchorage-dependent growth (ADG) of GEO cells. The 2 AR AS S-oligos were equipotent when used in equimolar concentrations. In particular, a 40% growth inhibition was observed at a concentration of 10 microM, while a mis-sense S-oligo used as control had no effect on GEO cell growth. The AR AS-1 S-oligo used at the same concentration also inhibited by 40% the 3H-thymidine incorporation by DNA of GEO cells. The anchorage-independent growth (AIG) of GEO cells was even more significantly affected by AR AS S-oligo treatment. In fact, up to 80% inhibition of the AIG of GEO cells was observed when cells were treated with 10 microM of both AR AS S-oligos. Finally, the AR AS S-oligos were able to specifically inhibit AR protein expression in GEO cells, as assessed by immunocytochemistry. These data suggest that AR is involved in colon-cancer cell transformation, and that AR may represent a suitable target for gene therapy in human colon carcinomas.

EGF-related peptides are involved in the proliferation and survival of MDA-MB-468 human breast carcinoma cells.

A majority of human breast carcinomas co-express the epidermal growth factor (EGF)-like peptides CRIPTO (CR), amphiregulin (AR) and transforming growth factor alpha (TGF-alpha). MDA-MB-468 breast carcinoma cells express CR, AR and TGFalpha, while SK-BR-3 cells express CR and TGF-alpha. Anti-sense phosphorothioate oligodeoxynucleotides (AS S-oligos) directed against either CR or TGF-alpha inhibit the proliferation of both cell lines. A 40-50% growth inhibition was observed at a 2-microM concentration of each AS S-oligo. Treatment with the AR AS S-oligo also resulted in a significant inhibition of MDA-MB-468 anchorage dependent growth (ADG). No significant growth inhibition was observed when MDA-MB-468 or SK-BR-3 cells were treated with a mis-sense S-oligo. The AS S-oligos inhibited the expression of AR, CR or TGF-alpha proteins and mRNAs, as assessed by immuno-cytochemistry and semi-quantitative RT-PCR. An additive growth-inhibitory effect was observed when MDA-MB-468 cells were treated with a combination of EGF-related AS S-oligos. Indeed, treatment of MDA-MB-468 cells with a combination of AR, CR and TGF-alpha AS S-oligos resulted in about 70% growth inhibition at a concentration of 0.7 microM each. Finally, treatment of MDA-MB-468 cells with a combination either of the 3 AS S-oligos or of an EGF receptor-blocking antibody (MAb 225) and either CR, AR or TGFalpha AS S-oligos resulted in a significant increase in DNA fragmentation. Our data suggest that the EGF-related peptides are involved in the proliferation and survival of breast carcinoma cells.