Department of Pharmacology and Therapeutics
Roswell Park Cancer Institute
Elm and Carlton Streets
Buffalo NY USA 14263
Tel: 716 - 845 - 5828
Fax: 716 - 845 - 8857
E-mail: bruce.dolnick@roswellpark.org

Program

rTS, signaling, Thymidylate synthase, acyl-homoserine lactones

Our laboratory is concerned with the role of the rTS gene in biology and cancer therapy. The rTS gene was discovered by our laboratory and was found to encode an antisense RNA to the key chemotherapy target thymidylate synthase (TS). In addition to producing an antisense RNA, the rTS gene encodes for the production of at least two proteins (rTSa, approximately 42 kDa; and the major protein product rTSâ, approximately 48 kDa). Expression of rTS is associated with growth arrest in cell culture, but its overexpression has been noted in cells resistant to anticancer drugs such as 5-fluorouracil and methotrexate. Expression of rTSâ is down-regulated in four major solid tumor types (breast, colon, kidney and lung) compared with normal tissue. Expression of rTSâ in cell culture is correlated with the production of signal molecules that cause the down-regulation of TS. A major objective of our laboratory (in collaboration with the laboratory of Dr. Janice Sufrin) is the isolation and chemical identification of these signal molecules, and the development of analogs that can be used to activate the rTS signaling pathway. In relation to this, our laboratory has developed a bioassay to screen for prospective compounds that can simulate the effects of rTS relevant to chemotherapy. Biological evaluation of the signal molecules produced by rTS-overexpressing cells has shown that these signaling molecules can cross-activate a receptor for acyl-homoserine lactones in a recombinant bacterial bioassay. The significance of this is that some opportunistic infectious organisms (such as Pseudomonas aeruginosa, which colonizes the lungs of immunocompromised cancer patients) may reciprocally utilize the rTS signaling pathway to overcome host defenses upon infection.

Progress

Development of a bioassay for thymidylate synthase modulators. Dr. Wu Qi in our laboratory has developed an assay that makes use of translational regulation through thymidylate synthase binding to an mRNA reporter. The assay is sensitive to compounds that alter thymidylate function or levels. This allows for high throughput screening for potential drugs that can down-regulate thymidylate synthase through the rTS signaling pathway.

Microarray studies of the effects of acyl-homoserine lactones have been used to determine the subsets of genes that are likely regulated by rTS signaling. Based upon the cross-activation of rTSâ-generated signals and acyl-homoserine lactones, we have used acyl-homoserine lactones to probe for a alterations in gene expression in a human colon tumor cell line. Genes with altered expression in response include functions of: nucleotide and folate metabolism, soluble ion transporters, cell-cyle arrest and quiescence, G protein signaling, sphingolipid metabolism.

Structure activity relationships of acyl-homserine lactone effects on cell growth and gene expression, conducted in collaboration with Dr. Janice Sufrin’s laboratory have revealed that optimal effects of these compounds correlate well with an acyl-side chain length of that produced by the infectious organism P. aeruginosa. In collaboration with Drs. J. McGuire and P. Liang, we have identified the existence of a mitochondrial form of rTSâ.

Select Publications

• Chu J, Dolnick BJ. Natural antisense (rTSa) RNA induces site-specific cleavage of thymidylate synthase mRNA. Biochemica et Biophysica Acta-Molecular Basis of Disease 1587:183-193, 2002.
• Wu Q, Dolnick BJ. Detection of thymidylate synthase modulators by a novel screening assay. Mol. Pharmacol. 63:167-173, 2003.
• Dolnick BJ, Angelino NJ, Dolnick R, Sufrin J.   A novel function for the rTS gene. Cancer Biology & Therapy 2(4):364-369, 2003.