Francis M. Sirotnak, PhD

The research of this laboratory focuses on genes regulating expression of molecular targets and other biochemical determinants important to:

• selective antitumor action of various categories of cytotoxic antimetabolites;
• cytoplasmic membrane transport of pharmacologic agents;
• molecular mechanisms of acquired resistance of tumor cells to antineoplastic agents; and
• the regulation of folate transporter gene expression.

Folates play a crucial role in the biosynthesis of macromolecules. Access of tumor cells to exogenous plasma folates is made possible by the existence in the cytoplasmic membrane of a specific high-affinity transport system encoded by the RFC-1 gene.

Using cDNA probes, the genetic regulation and molecular genetics of this system are now being examined in models that constitutively overproduce or underproduce the transport protein and during induction of tumor cells to terminal maturation. The organization and structure of this gene and its mRNA splicing, and the characteristics of the promoters regulating its transcription, are also under study.

Recent evidence from this laboratory has shown that oncogene expression upregulates RFC-1 expression at the level of transcription following neoplastic transformation. Thus RFC-1 is a target gene of these oncogenes. Studies are focusing on the molecular basis for this relationship and the manner in which these findings can be exploited to improve the responsiveness of tumors to folate analogues.

Folate analogs effectively accumulate in tumor cells via plasma membrane systems normally transporting natural folates. To understand the selective antitumor action of folate analogues, studies are being conducted in tumor cells on:

• the properties and multiplicity of their cellular membrane transport mechanisms;
• their interaction with enzyme and macromolecular targets;
• their intracellular metabolic disposition; and
• their pharmacokinetic behavior.

Mechanisms of acquired resistance in tumor cells to these antimetabolites and other cytotoxic agents are also studied at the level of the relevant genes that control these activities. Of special emphasis are our studies on the folate polyglutamylation enzyme -- folylpolyglutamate synthetase -- which include investigations of the regulation of its gene expression with appropriate molecular probes. The structure of this gene is also under study as well as the promoters that regulate its transcription and tissue-specific mRNA splicing.

Other studies utilize structural-based design of new folate analogues of the 10-deazaaminopterin series as therapy for neoplastic disease. This design feature exploits quantitative structure-activity assessment of these analogues at sites of membrane transport and intracellular metabolism that are identified as determinants of therapeutic response.

A newly designed analogue -- 10-propargyl-10-deazaaminopterin is now under study, in Phase II clinical trials, on the basis of preclinical studies against human tumor xenografts that documented marked superiority over methotrexate.