Kevin J. Scanlon

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.

Hammerhead ribozyme against γ-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 γ-glutamylcysteine synthetase (γ-GCS), a rate-limiting enzyme for GSH. We constructed a hammerhead ribozyme against a γ-GCS heavy subunit (γ-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–γ-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. Cancer Gene Therapy (2001) 8, 803–814

Oligonucleotides as modulators of cancer gene expression

The delineation of gene function has always been an intensive subject of investigations. Recent advances in the synthesis and chemistry of oligonucleotides have now made these molecules important tools to study and identify gene function and regulation. Modulation of gene expression using oligonucleotides has been targeted at different levels of the cellular machinery. Triplex forming oligonucleotides, as well as peptide nucleic acids, have been used to inhibit gene expression at the level of transcription; after binding of these specific oligonucleotides, conformational change of the DNAs helical structure prevents any further DNA/protein interactions necessary for efficient transcription. Gene regulation can also be achieved by targeting the translation of mRNAs. Antisense oligonucleotides have been used to down-regulate mRNA expression by annealing to specific and determined region of an mRNA, thus inhibiting its translation by the cellular machinery. The exact mechanism of this type of inhibition is still under intense investigation and is thought to be related to the activation of RNase H, a ribonuclease that is widely available that can cleave the RNA/DNA duplex, thus making it inactive. Another well-characterized means of interfering with the translation of mRNAs is the use of ribozymes. Ribozymes are small catalytic RNAs that possess both site specificity and cleavage capability for an mRNA substrate, inhibiting any further protein formation. This review describes how these different oligonucleotides can be used to define gene function and discusses in detail their chemical structure, mechanism of action, advantages and disadvantages, and their applications.

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.

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.

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.

Ribozyme‐Mediated Cancer Gene Therapy

Certain RNA molecules, called ribozymes, possess site-specific cleavage activities. Ribozymes were initially discovered as the intervening sequence of the RNA Group I intron which catalyzes its own excision, and this has been called self-splicing.J Several naturally occurring ribozymes, such as RNase P, Group I1 intron, hammerhead ribozymes, hairpin ribozymes, and hepatitis delta virus ribozymes have also been identified.3,4 Although ribozymes were originally found to act through a cis reaction, they have been engineered to cleave their targets in trans in a truly catalytic manner.j Due to their catalytic potential, ribozymes have been investigated as agents to block genetic information. A crucial role of ribozymes as therapeutic agents would be to downregulate a specific gene by interfering with the information from the nucleic acid to the protein, i.e. mRNA. Hammerhead ribozymes have demonstrated the ability to inhibit the expression of specific genes by targeting their mRNAs.6 Because of their small structure, simple cleavage mechanism, and specificity, hammerhead ribozymes have been extensively investigated for potential therapeutic application^.^^^ In principle, hammerhead ribozymes can be designed to target mRNAs of any disease process in which a specific protein has been linked to its etiology. Certain diseases including neoplastic diseases are often caused by the undesirable expression of mRNA, and are thought to be appropriate targets for ribozymes. Since cancer is a genetic disease, gene targeted therapy could be a promising strategy for the treatment of specific cancers. Extensive research has helped clarify the mechanism of tumorigenesis, tumor progression, and metastatic processes. Several oncogenes and tumor suppressor genes have been identified and linked with specific ma l ignanc ie~ .~ -~~ Although multistep processes of cancer progression have been demonstrated and several genes are thought to be involved, the elimination of a single

Mechanisms of Cisplatin Resistance and Its Reversal In Human Tumors

Despite tremendous strides in understanding the molecular basis of cancer (Weinberg, 1989), treatment of human cancer is still limited by the toxicity of chemotherapeutic agents and the development of intrinsic or acquired resistance to these drugs. cis-diamminedichloroplatinum (II) (cisplatin) is one of the most widely-used anticancer agents, active in the treatment of ovarian, testicular, head-and-neck, non-small cell lung and brain tumors, among others (Rosenberg, 1985). However, the rapid development of resistance to cisplatin represents an important challenge to clinicians and laboratory investigators alike. Therefore, understanding the biochemical and molecular basis of cisplatin resistance may potentially result in the development of rational approaches to circumvent this problem. At the core of understanding cisplatin resistance lies the realization of both the similarities and differences between the mechanisms of cisplatin action and resistance and that of other chemotherapeutic agents. Cisplatin-resistant cells display a unique cross-resistance pattern to multiple agents, including anti-metabolites such as 5-fluorouracil and methotrexate, DNA polymerase inhibitors such as azidothymidine (AZT), and topoisomerase inhibitors such as camptothecin and etoposide. This “atypical” multidrug resistance is both phenotypically and molecularly distinct from the “classical” multidrug resistance which may involve overexpression of the MDR-1 gene (Gottesman and Pastan, 1993).

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.

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. Cancer Gene Therapy (2001) 8, 906–908