Cisplatin, carboplatin and oxaliplatin are platinum-based drugs that are widely used in cancer chemotherapy. Platinum–DNA adducts, which are formed following uptake of the drug into the nucleus of cells, activate several cellular processes that mediate the cytotoxicity of these platinum drugs. This review focuses on recently discovered cellular pathways that are activated in response to cisplatin, including those involved in regulating drug uptake, the signalling of DNA damage, cell-cycle checkpoints and arrest, DNA repair and cell death. Such knowledge of the cellular processing of cisplatin adducts with DNA provides valuable clues for the rational design of more efficient platinum-based drugs as well as the development of new therapeutic strategies.

Cellular processing of platinum anticancer drugs.

Structure, Recognition, and Processing of Cisplatin-DNA Adducts.

A method for the quantitation of specific mRNA species by the polymerase chain reaction (PCR) has been developed by using a synthetic RNA as an internal standard. The specific target mRNA and the internal standard are coamplified in one reaction in which the same primers are used. The amount of mRNA is then quantitated by extrapolating against the standard curve generated with the internal standard. The synthetic internal standard RNA consists of a linear array of the sequences of upstream primers of multiple target genes followed by the complementary sequences to their downstream primers in the same order. This quantitative PCR method provides a rapid and reliable way to quantify the amount of a specific mRNA in a sample of less than 0.1 ng of total RNA. In addition, the same internal standard RNA is used, with appropriate primer pairs, to quantitate multiple different mRNA species.

https://www.pnas.org/content/pnas/86/24/9717.full.pdf

Quantitation of mRNA by the Polymerase Chain Reaction

Molecular mechanisms of resistance and toxicity associated with platinating agents.

Extensive research over the past 30 years has revealed the involvement of Ras not only in tumorigenesis but also in many developmental disorders. The complexity of the molecular and cell biological mechanisms of action of Ras proteins indicates that much remains to be learnt.

Ras oncogenes: split personalities

The c-fos gene product Fos has been implicated in many cellular processes, including signal transduction, DNA synthesis, and resistance to antineoplastic agents. A fos ribozyme (catalytic RNA) was designed to evaluate the effects of suppressing Fos protein synthesis on expression of enzymes involved in DNA synthesis, DNA repair, and drug resistance. DNA encoding the fos ribozyme (fosRb) was cloned into the pMAMneo expression plasmid, and the resultant vector was transfected into A2780DDP cells resistant to the chemotherapeutic agent cisplatin. The parental drug-sensitive A2780S cells were transfected with the pMMV vector containing the c-fos gene. Morphological alterations were accompanied by significant changes in pharmacological sensitivity in both c-fos- and fosRb-transfected cells. pMAMneo fosRb transfectants revealed decreased c-fos gene expression, concomitant with reduced thymidylate (dTMP) synthase, DNA polymerase beta, topoisomerase I, and metallothionein IIA mRNAs. In contrast, c-myc expression was elevated after fos ribozyme action. Insertion of a mutant ribozyme, mainly capable of antisense activity, into A2780DDP cells resulted in smaller reductions in c-fos gene expression and in cisplatin resistance than the active ribozyme. These studies establish a role for c-fos in drug resistance and in mediating DNA synthesis and repair processes by modulating expression of genes such as dTMP synthase, DNA polymerase beta, and topoisomerase I. These studies also suggest the utility of ribozymes in the analysis of cellular gene expression.

https://www.jstor.org/stable/pdfplus/2358296.pdf

Ribozyme-mediated cleavage of c-fos mRNA reduces gene expression of DNA synthesis enzymes and metallothionein

In this study, the efficacy of an anti- ras ribozyme in reversing the neoplastic phenotype was investigated. Murine NIH3T3 cells were trans-fected with cellular DNA from the FEMX-I human melanoma cell line expressing the activated H- ras gene. The transformed cells displayed the neoplastic phenotype in vitro and were tumorigenic in nude mice in vivo . When the transformants were transfected by a ribozyme designed to cleave only activated H- ras RNA, the transformed phenotype was abrogated. In contrast, expression of a mutant ribozyme, essentially acting only as antisense, into the transformed cells resulted in less dramatic changes in cell growth and tumorigenicity. These results reinforce the potential role of anti-oncogene ribozymes as suppressors of neoplastic growth, with possible implications for gene therapy.

/pdf/suppression-of-the-neoplastic-phenotype-in-vivo-by-an-anti-gf3ewvxor3.pdf

Suppression of the Neoplastic Phenotype in Vivo by an Anti-ras Ribozyme

Strategies have been developed to abrogate the aberrant expression of dominant oncogenes as a means to accomplish targeted tumor eradication. We have demonstrated previously the utility of this approach using a hammerhead ribozyme designed to cleave the mutant sequence in codon 12 of the activated H-ras oncogene transcript. To develop this strategy into a practical means to approach malignant disease, methods must be developed to accomplish high efficiency delivery of the ribozyme to target neoplastic cells. To accomplish this, a recombinant adenovirus was designed that encoded a gene cassette for the H-ras ribozyme. By using this virus, it was possible to accomplish high efficiency reversion of the neoplastic phenotype in mutant H-ras expressing tumor cells without the need for any selection steps. The demonstration of the utility of adenoviral-mediated delivery of anticancer ribozymes will allow the practical development of gene therapy strategies on this basis.

https://cancerres.aacrjournals.org/content/canres/55/10/2024.full.pdf

Neoplastic Reversion Accomplished by High Efficiency Adenoviral-mediated Delivery of an Anti-ras Ribozyme

In this review, cisplatin resistance will be summarized. We will discuss characteristics of potential mechanisms of resistance in tissues from patients with ovarian, colon and breast carcinoma. Secondly, a new molecular biology assay will be discussed which detects changes in gene expression in cisplatin-resistant cells. Finally, based on the biochemical and molecular properties of cisplatin-resistant tumors, new strategies to exploit or circumvent cisplatin resistance in human tumors will be entertained.

Molecular basis of cisplatin resistance in human carcinomas: model systems and patients.

The notion that oligonucleotides can modulate gene-specific expression was established more than a decade ago. Recent advances in molecular genetics have broadened the armamentarium used to manipulate gene expression in biological systems including triplex DNA, antisense RNA/DNA, and ribozymes (catalytic RNA). These oligonucleotides demonstrated important early application to the elucidation of cellular signaling pathways. More recently, studies with these agents have probed their utility as potential therapeutic agents, especially in the realm of cancer. With the implementation of gene therapy in early clinical trials, oligonucleotide-mediated suppression of gene expression has emerged as an important strategy for gene therapy. This review will discuss the current knowledge in this field, focusing on the biology of triplex DNA, antisense oligonucleotides, and ribozymes.

https://faseb.onlinelibrary.wiley.com/doi/pdf/10.1096/fasebj.9.13.7557018

Kevin J. Scanlon

Papers

Ribozyme-mediated cleavage of c-fos mRNA reduces gene expression of DNA synthesis enzymes and metallothionein

Suppression of the Neoplastic Phenotype in Vivo by an Anti-ras Ribozyme

Neoplastic Reversion Accomplished by High Efficiency Adenoviral-mediated Delivery of an Anti-ras Ribozyme

Molecular basis of cisplatin resistance in human carcinomas: model systems and patients.

Oligonucleotide-mediated modulation of mammalian gene expression.