Growth inhibition efficacy of an adenovirus expressing dual therapeutic genes, wild-type p53, and anti-erbB2 ribozyme, against human bladder cancer cells.

The altered expression of both p53 and erbB2 is strongly related to the disease status and the outcome of bladder cancers. We examined the antitumor efficacy by the modulation of these genetic alterations with a newly designed dual-gene-expressing adenovirus (Ad-p53/erbB2Rz), which expresses p53 and anti-erbB2 ribozyme simultaneously in human bladder cancer cells. Cell growth inhibition efficacy along with biological responses of this virus was compared with other viral vectors (Ad-p53, which expresses wild-type p53 cDNA, and Ad-erbB2Rz, which expresses anti-erbB2 ribozyme, solely or in combination). Sufficient transgene expression in targeted cells and the altered expression of the targeted genes and their encoded proteins were obtained by each therapeutic vector. Each of the three therapeutic viral vectors inhibited bladder cancer cell growth, and the putative additive antitumor effect was shown by the combination of two of the therapeutic vectors. Furthermore, Ad-p53/erbB2Rz had superior therapeutic efficacy when the same titers of viruses were infected. Nonspecific vector-related toxicity was minimized by reducing the total amount of viral titers by using the dual-gene-expressing adenovirus. Modulation of multiple genetic abnormalities might enhance the therapeutic efficacy, and vector-related toxicity could be minimized when the total amount of viral titers are reduced.

Hammerhead ribozyme against gamma-glutamylcysteine synthetase sensitizes human colonic cancer cells to cisplatin by down-regulating both the glutathione synthesis and the expression of multidrug resistance proteins.

Multidrug resistance in cancer cells is often associated with an elevation in the concentration of glutathione (GSH) and the expression of gamma-glutamylcysteine synthetase (gamma-GCS), a rate-limiting enzyme for GSH. We constructed a hammerhead ribozyme against a gamma-GCS heavy subunit (gamma-GCSh) mRNA transcript and transfected it to human colonic cancer cells (HCT8DDP) resistant to cisplatin (CDDP). The effect of the ribozyme transfection on the drug resistance of cancer cells was studied. (a) Transfection of the ribozyme decreased the GSH level and the efflux of CDDP-GSH adduct, resulting in higher sensitivity of the cells to CDDP. (b) The transfection suppressed the expression of ATP-binding cassette (ABC) family of transporters such as MRP1, MRP2, and MDR1, and stimulated the expression of mutant p53. (c) An electrophoretic mobility shift assay showed that mutant p53 suppresses the SP1-DNA binding activity, suggesting that this mutant p53 is functional and it, in turn, suppresses the expression of ABC transporters. Collectively, transfection of anti-gamma-GCSh ribozyme reduced the synthesis of GSH and the expression of ABC transporters, which causes an increase in the sensitivity of cancer cells to anticancer drugs. Suppression of the SP1-DNA binding activity by p53 may be a factor of down-regulation of ABC transporters.

Reversal of cisplatin and multidrug resistance by ribozyme-mediated glutathione suppression.

gamma-Glutamylcysteine synthetase (gamma-GCS) is a key enzyme in glutathione (GSH) synthesis, and is thought to play a significant role in intracellular detoxification, especially of anticancer drugs. Increased levels of GSH are commonly found in the drug-resistant human cancer cells. We designed a hammerhead ribozyme against gamma-GCS mRNA (anti-gamma-GCS Rz), which specifically down-regulated gamma-GCS gene expression in the HCT-8 human colon cancer cell line. The aim of this study was to reverse the cisplatin and multidrug resistance for anticancer drugs. The cisplatin-resistant HCT-8 cells (HCT-8DDP cells) overexpressed MRP and MDR1 genes, and showed resistance to not only cisplatin (CDDP), but also doxorubicin (DOX) and etoposide (VP-16). We transfected a vector expressing anti-gamma-GCS Rz into the HCT-8DDP cells (HCT-8DDP/Rz). The anti-gamma-GCS Rz significantly suppressed MRP and MDR, and altered anticancer drug resistance. The HCT-8DDP/Rz cells were more sensitive to CDDP, DOX and VP-16 by 1.8-, 4.9-, and 1.5-fold, respectively, compared to HCT-8DDP cells. The anti-gamma-GCS Rz significantly down-regulated gamma-GCS gene expression as well as MRP/MDR1 expression, and reversed resistance to CDDP, DOX and VP-16. These results suggested that gamma-GCS plays an important role in both cisplatin and multidrug resistance in human cancer cells.

Modulation of multidrug resistance in a cancer cell line by anti-multidrug resistance-associated protein (MRP) ribozyme.

Multidrug resistance-associated protein (MRP) is the major candidate molecule responsible for non-P-glycoprotein (PGp)-mediated multidrug resistance. We used a hammerhead anti-MRP ribozyme (alpha MRP-Rz) to inactivate MRP function in a multidrug resistant cancer cell line, KB8-5. The beta-actin promoter-driven alpha MRP-Rz sequence (pH beta/alpha MRP-Rz) was introduced into KB8-5 cells (KB8-5/alpha MRP-Rz) and we evaluated growth of the cell line. The gene expression of multidrug resistance-related molecules was estimated. Drug sensitivity was estimated by MTT assay in vitro. MRP mRNA expression was decreased in KB8-5/alpha MRP-Rz cells. The MTT assay showed increased IC50 values or resistance to doxorubicin (DOX), etoposide (VP-16) and cisplatin (CDDP) in KB8-5/alpha MRP-Rz cells. No significant differences were observed in expression of multidrug resistance gene (MDR1), thymidylate synthase, glutathione S-transferase pi or topoisomerase II alpha. The hammerhead ribozyme-mediated simple suppression of MRP mRNA expression was not sufficient to reverse multidrug resistance in the cancer cell line KB8-5.

An overview of the Tenth International Conference on Cancer Gene Therapy.

Dr. Scanlon, the new president (2001-2002) of the International Society of Cancer Gene Therapy, gave the conference summary and an overview of gene therapy in the new millennium. The conference reflected the progress made in the development of new promoters and improved delivery systems for gene therapy. Many presentations and posters focused on the progress in these areas. These scientific findings in the field of gene therapy may ultimately be exploited in the future developments of stem cell research. Conversely, challenges still remain before gene therapy will significantly impact cancer. The basic science of the cancer model systems lacked the ability to reflect the clinical reality of patient treatment. This places the burden on the physicians to be more vigilant to subtle changes in patient response that were not observed in the preclinical models. The bystander effect has yet to be fully understood and needs further clinical validation. Systemic delivery needs to be further addressed before a marketable product can be developed. The delivery systems discussed at the conference lack the ability to achieve pharmacological doses of therapeutic genes in the target tissue. Until these challenges are addressed, gene therapy will remain on the sidelines as a cancer modality. Yet, the field should be optimistic with the current progress. The future influences of the digital and genomic revolution in the health care industry will certainly impact the design of products for gene therapy. Dr. Scanlon concluded that the education of scientists would cross over into diverse disciplines so that novel observations will be exploited for new therapies. This gene therapy series will continue with the International Conference on the Gene Therapy of Cancer, scheduled for December 13-15, 2001, in San Diego, CA.

Ribozyme as an approach for growth suppression of human pancreatic cancer.

Ribozymes (catalytic RNAs, RNA enzymes) are effective modulators of gene expression because of their simple structure, site-specific cleavage activity, and catalytic potential, and have potentially important implications for cancer gene therapy. Point mutations in the K-ras oncogene are found in approx 90% of human pancreatic carcinomas, and can be used as potential targets for specific ribozyme-mediated reversal of the malignant phenotype. In this study, we focused on in vitro manipulation of ribozyme targeting of the mutated K-ras oncogene in a human pancreatic carcinoma cell line. We evaluated the efficacy of an anti-K-ras hammerhead ribozyme targeted against GUU-mutated codon 12 of the K-ras gene in cultured pancreatic carcinoma cell lines. The anti-K-ras ribozyme significantly reduced cellular K-ras mRNA level (GUU-mutated codon 12) when the ribozyme was transfected into the Capan-1 pancreatic carcinoma cells. The ribozyme inhibited proliferation of the transfected Capan-1 cells. These results suggested that this ribozyme is capable of reversing the malignant phenotype in human pancreatic carcinoma cells.

Adenovirus-mediated anti-K-ras ribozyme induces apoptosis and growth suppression of human pancreatic carcinoma.

Human pancreatic cancer is a lethal malignancy, and the lesions show a very high incidence of point mutations of the K-ras oncogene. These alterations can be used as potential targets for specific ribozyme (Rz)-mediated growth suppression of the cancer cells. We designed an anti-K-ras Rz against mutant K-ras gene transcripts (codon 12, GGT to GTT) and generated a recombinant adenovirus (rAd) to express the Rz (rAd/anti-K-ras Rz). More than 95% of Capan-1 human pancreatic cells were infected with rAd/anti-K-ras Rz when treated with the virus at 200 plaque-forming units/cell. The virus, rAd/anti-K-ras Rz, significantly suppressed mutant K-ras gene expression and inhibited the growth of Capan-1 cells. At 3 days postinfection, we observed maximum growth suppression of the cells, characteristic morphological changes of apoptosis such as nuclear condensation and oligonucleosomal DNA fragmentation, and suppression of bcl-2 oncoprotein. These changes were not found in control virus-infected cells. Our results indicated that the virus rAd/anti-K-ras Rz specifically down-regulated the K-ras/bcl-2 pathway and induced apoptotic changes in Capan-1 pancreatic carcinoma cells. High-efficiency adenovirus-mediated delivery of anti-K-ras Rz could become a significant gene therapy strategy against human pancreatic cancer.

Anti-K-ras ribozyme induces growth inhibition and increased chemosensitivity in human colon cancer cells.

Colon cancer is one of the carcinomas that is resistant to a variety of therapies. To develop a new therapy by regulating the activated K-ras gene in colon cancers, we prepared synthetic DNA encoding the ribozyme (catalytic RNA), and inserted it into an expression vector (LNCX) originated from a retrovirus. Transfection of the vector into SW620 human colon cancer cells brought about significant suppression of K-ras mRNA synthesis and inhibition of the cell growth. Studies in athymic mice, in which K-ras ribozyme-introduced SW620 cells were transplanted, also revealed a marked reduction of tumor growth in vivo. Furthermore, the ribozyme-introduced tumors became more sensitive to treatment with anti-cancer drugs such as cisplatin, adriamycin, 5-fluorouracil, vincristine, and etoposide. These data suggest that the possible efficacy of anti-K-ras ribozyme increases the chemosensitivity of human colon cancers as well as the inhibitory effect on the growth of human colon cancers.

Adenovirus-mediated ribozyme targeting of HER-2/neu inhibits in vivo growth of breast cancer cells.

HER-2/neu is overexpressed in 25-30% of human breast cancers. We prepared an anti-HER-2/neu hammerhead ribozyme expressed by a recombinant adenovirus (rAdHER-Rz). Human breast cancer cell lines were transduced with high efficiency, resulting in decreased HER-2/neu expression. In vivo injections of rAdHER-Rz into BT-474 tumors established in nude mice inhibited tumor growth to 20% of mock-treated controls. Similar in vivo effects were shown in MCF-7 cells, which do not overexpress HER-2/neu. The growth inhibitory effects of rAdHER-Rz were greater than those of an antisense-expressing vector. These results suggest the utility of anti-HER-2/neu ribozymes as a rational strategy for gene therapy of breast cancer. Gene Therapy (2000) 7, 241-248.

Anti-tumorigenic effect of a K-ras ribozyme against human lung cancer cell line heterotransplants in nude mice.

Approximately 15-30% of human non-small cell lung cancers (NSCLC) carry K-ras mutations, among which point mutations at codon 12 are the most common. This study characterizes the anti-tumor effect of an anti-K-ras ribozyme adenoviral vector (KRbz-ADV; replication-deficient, E1-deleted Ad5 backbone) against NSCLC lines that express the relevant mutation (K-ras codon 12 GGT --> GTT; H441 and H1725). KRbz-ADV significantly inhibited tumor cell growth (38-94% reduction by 3H-thymidine uptake) in a time- and dose-dependent manner, but produced minimal growth inhibition on normal epithelial cells, or NSCLC H1650 cells that lack the relevant mutation. The in vivo anti-tumorigenic effect of KRbz-ADV treatment was characterized with cell line xenografts in nu/nu mice. Pre-treatment with KRbz-ADV (10 or 20 p.f.u. per cell) completely abrogated subcutaneous engraftment of H441 (n = 13) or H1725 cells (n = 8), as compared with a 100% tumor take and progressive tumor growth in animals that received untreated tumor cells, or control vector (luciferase-adenovirus/Luc-ADV)-treated tumor cells. Pre-treatment with a mutant anti-K-ras ribozyme adenoviral vector (mutKRbz-ADV), which has the same specificity as KRbz but lacks ribozyme catalytic activity, did not produce an anti-tumorigenic effect. The in vivo effect of KRbz-ADV treatment was further examined by initiating injections (2 x 10(9) p.f.u.) at 7 days after tumor induction. Pre-existing tumor growth was reduced by 39% by a single intratumoral injection. Repeat injections (three or five KRbz-ADV-intratumoral injections at 2 x 10(9) p.f.u. every other day) resulted in complete tumor regression in five of seven mice. In contrast, single or multiple injections of control vector Luc-ADV did not significantly alter tumor xenograft outcome. Ribozyme expression was confirmed in H441 cells that demonstrated reduced growth after KRbz-ADV treatment. Reduced growth corresponded to significantly lowered levels of K-ras mRNA, as defined by RT-PCR (51% of untreated level, n = 3) and RNase protection assay (56% of untreated level, n = 4) analyses. Further, 37.5% of KRbz-ADV-treated cells underwent apoptosis, as compared with 11.7%, and 19.0% in untreated and Luc-ADV-treated cultures, respectively. A significantly higher proportion of KRbz-ADV-treated H441 cells (58.2%) underwent apoptosis when maintained under anchor-independent conditions that simulate in vivo tumorigenesis ('anoikis'). This is the first report that demonstrates that KRbz-ADV can effectively inhibit in vivo tumorigenesis, and produces regression of pre-existing human lung tumor xenografts having the relevant K-ras mutation.

Hammerhead ribozymes as therapeutic agents for bladder cancer.

Hammerhead ribozymes have been investigated extensively as therapeutic agents against cancer. Aberrant or overexpression of genes related to tumorigenicity or cancer growth might be the appropriate targets for ribozyme strategies. Ribozyme-mediated gene therapy should be applied to those diseases that have no successful conventional therapy such as advanced or treatment-resistant bladder cancer. Many genetic alterations have been identified in bladder cancer related to both tumorigenesis and disease progression. Mutated H-ras, fos, and erb-B2 genes have been chosen as targets for ribozymes in previous studies, and antitumor efficacy has been demonstrated by reversion of the malignant phenotypes and by inhibition of tumor growth both in vitro and in vivo. The efficiency of various delivery systems has also been evaluated. An overview of ribozyme strategies, especially for therapeutic applications against bladder cancer, is described here.

Anti-c-erb1B2 Ribozyme for Gene Therapy of Breast Cancer.

The application of antioncogene ribozyme in the gene therapy of breast cancer by means of recombinant adenoviral vector is dicussed in this chapter. We have shown that recombinant adenovirus encoding anti-cerbB2 ribozyme inhibited the breast cancer cell growth in vivo efficiently (1,2). We will talk about the detailed protocol here.

Therapeutic efficacy of an adenovirus-mediated anti-H-ras ribozyme in experimental bladder cancer.

Ras oncogenes are thought to play a critical role in cellular proliferation and tumorigenesis. Reversal of the malignant phenotype, inhibition of tumor growth, and decreased tumorgenicity have been demonstrated with the use of anti-H-ras ribozymes. In this study, the therapeutic efficacy of a hammerhead ribozyme targeting the mutated H-ras oncogene was investigated in an experimental bladder cancer model using a recombinant adenovirus as delivery vehicle. Tumors were established in nude mice by subcutaneous injection of EJ human bladder carcinoma cells harboring a point mutation of the H-ras gene. The tumors were treated with intralesional injections of an adenovirus expressing an anti-H-ras ribozyme (rAd-Hras Rz) by different schedules at serial titers, and the tumor inhibition efficacy was analyzed. The viral infection efficacy and kinetics of ribozyme expression were also evaluated. Intralesional injection of rAd-Hras Rz resulted in significant antineoplastic effects in a dose-dependent fashion. Complete regression of the tumor was achieved by rAd-Hras Rz in several cases without recurrence during the 50-day observation period. Although there was moderate vector-associated cytotoxicity in this cell line, complete regressions were not observed in the cases treated with control adenovirus vectors or vectors expressing an inactive anti-H-ras ribozyme or anti-H-ras antisense oligonucleotides. These results suggest the efficacy of a ribozyme-encoding adenovirus in the experimental gene therapy of human bladder cancer.

Altered expression of mRNAs for apoptosis-modulating proteins in a low level multidrug resistant variant of a human lung carcinoma cell line that also expresses mdr1 mRNA.

An in vitro model that might be relevant to cancer cell chemoresistance in vivo was generated by exposing the human lung carcinoma clonal cell line DLKP-SQ to 10 sequential pulses of pharmacologically attainable doses of doxorubicin. The resistant variant, DLKP-SQ/10p, was found to be cross-resistant to doxorubicin (10x), vincristine (43x), etoposide (3x), sodium arsenate (3x), paclitaxel (38x) [which could imply overexpression of P-glycoprotein (P-gp) and possibly increased multidrug resistance-associated protein activity] and 5-fluorouracil (4x), but slightly sensitized to carboplatin. Analysis of mRNA levels in the resistant variant revealed overexpression of mdr1 mRNA without significant alteration in mrp, Topo. IIalpha, GSTpi, dhfr or thymidylate synthase mRNA levels. Overexpression of the anti-apoptotic bcl-xL transcript and the pro-apoptotic bax mRNA was also detected but no alterations in bcl-2 or bag-1 mRNA levels were observed. Resistance to a P-gp-associated drug, doxorubicin, could be reversed with P-gp circumventing agents such as cyclosporin A and verapamil, but these substances had no effect on resistance to 5-fluorouracil. Overexpression of the pro-apoptotic bcl-xS gene in the DLKP-SQ/10p line partially reversed resistance not only to P-gp-associated drugs but also to 5-fluorouracil, indicating that the ratio of bcl family members may be important in determining sensitivity to chemotherapeutic drug-induced apoptosis.

Hammerhead ribozymes against gamma-glutamylcysteine synthetase mRNA down-regulate intracellular glutathione concentration of mouse islet cells.

gamma-Glutamylcysteine synthetase (gamma-GCS) is a key enzyme in glutathione synthesis and is thought to play a significant role in intracellular detoxification systems. To specifically suppress gamma-GCS gene expression, we constructed two different hammerhead ribozymes against gamma-GCS mRNA transcripts. Two cleavage sites were targeted as follows: site 1 for anti-gamma-GCS ribozyme (H), a GUU triplet located from +348 to +350 of the gamma-GCS heavy chain, and site 2 for anti-gamma-GCS ribozyme (L), a GUU triplet located from +235 to +237 of the gamma-GCS light chain. The anti-gamma-GCS ribozymes effectively cleaved gamma-GCS mRNA in a cell-free system. When transfected into a Min-6 mouse islet cell line, these anti-gamma-GCS ribozymes not only suppressed gamma-GCS gene expression, but also reduced intracellular glutathione concentration. These results suggest that the ribozyme-mediated down-regulation of gamma-GCS gene expression may be useful for analyzing the glutathione-associated cellular defense systems of pancreatic islet cells.

Hammerhead ribozyme specifically inhibits mutant K-ras mRNA of human pancreatic cancer cells.

We have evaluated the efficacy of an anti-K-ras hammerhead ribozyme targeted against GUU-mutated codon 12 of the K-ras gene in a cell-free system as well as in cultured pancreatic carcinoma cell lines. In the cell-free system, the anti-K-ras ribozyme specifically cleaved K-ras RNA with GUU-mutation at codon 12, but not other triplet sequences at codon 12 of K-ras RNA. In the cell culture system, the anti-K-ras ribozyme significantly reduced K-ras mRNA level (GUU-mutated codon 12) in Capan-1 pancreatic carcinoma cells, but less significantly suppressed K-ras mRNA in Capan-2 (GUU/GGU heterozygous-mutation at codon 12) or MIA PaCa-2 (UGU-mutated codon 12) pancreatic carcinoma cells. The ribozyme inhibited proliferation of transfected Capan-1 cells. These results suggest that this ribozyme selectively recognizes single-base mutation of K-ras mRNA and is able to reverse the malignant phenotype in human pancreatic carcinoma cells.

Reversion of multidrug resistance in the P-glycoprotein positive breast cancer cell line (MCF-7/ADR) by introduction of hammerhead ribozyme.

A hammerhead ribozyme which site-specifically cleaved the GUC position in codon 880 of the mdr1 mRNA was designed. The target site was chosen between the two ATP binding sites, which may be important for the function of the P-Gp as an ATP-dependent pump. A DNA sequence encoding the ribozyme gene was then incorporated into a eukaryotic expression vector (pH beta Apr-1 neo) and transfected into the breast cancer cell line MCF-7/Adr, which is resistant to adriamycin and expresses the MDR phenotype. The ribozyme was stably expressed in the cell line by the RNA dot blotting assay. The result of Northern blot assay showed that the expressed ribozyme could decrease the level of mdr1 mRNA expression by 83.5%; and the expressed ribozyme could inhibit the formation of P-glycoprotein detected by immuno-cytochemistry assay and could reduce the cell's resistance to adriamycin; this means that the resistant cells were 1,000-fold more resistant than the parental cell line (MCF-7), whereas those cell clones that showed ribozyme expression were only 6-fold more resistant than the parental cell line. These results show that a potentially useful tool is at hand which may inactivate MDR1 mRNA and revert the multidrug resistance phenotype.