Cloning and characterization of a naturally occurring antisense RNA to human thymidylate synthase mRNA

Abstract

Based upon reverse transcription and polymerase chain reaction results with human KB cell RNA, a cDNA (i.e., 3'rTS1, 1557 nt) with complementarity to thymidylate synthase mRNA was cloned and sequenced. Northern blot analysis showed that 3'rTS1 corresponded to a cytoplasmic 1.8 kb RNA found in several tumor cell lines. The remaining 5' region of this antisense RNA was cloned by a RACE (Rapid Amplification of cDNA Ends) procedure. A full length cDNA (i.e., rTS, 1811 nt) was generated by splicing 3'rTS1 with RACE-generated cDNA. rTS RNA is likely a mRNA that contains four open reading frames. Based upon sequence analysis of the RACE cDNAs and the rTS cDNA, rTS RNA is likely processed from a gene containing at least six introns. Northern blot analysis indicates rTS RNA is expressed in a variety of human tumor cell lines and an aberrant form is expressed in a methotrexate-cell line.

Expression of rTSβ as a 5-fluorouracil resistance marker in patients with primary breast cancer

“…These results, however, did not fulfill the expected enzyme function. On the other hand, when considered together with clinical outcomes, our work suggested that the nuclear level of rTSß may not only be associated with increased 5-FU resistance, but also with a higher incidence of lymph node involvement, ER and/or PR expression, Her2-neu gene amplification and advanced tumor stage, events that are intimately associated with the rapid growth of breast cancer cells (14)(15)(16), and that rTSß may act as a nucleus-housed glycolytic protein, which may play a role during G2/M phases of cell cycle progression.…”

“…Treatment with manumycin A, on the other hand, decreased expression and nuclear translocation of rTSß. The addition of manumycin A also inhibits the protein level of TS and α-tubulin, and such results suggest that farnesylation of rTSß could prevent protein degradation and maintain protein level in the cells (14)(15)(16). Moreover, our data indicated that like fatty acyl modification of the G-proteins, which help in determining the final destination of the protein either to the plasma membrane or to the endoplasmic reticulum to carry out the respective biological functions (32), farnesylation could also help in determining the organelle targeting of rTSß.…”

“…The biological presentation of rTS, which is located on the same stretch of chromosome, but in the opposite direction to the TS gene, has suggested its characteristics in counteracting TS gene expression (14). An in vitro study by Dolnick et al showed that cultured tumor cells with elevated rTS gene expression are frequently associated with increased resistance to 5-FU and methotrexate (MTX) (15).…”

“…Breast cancer cells, 60055, BT-20, MCF-1, MCF-7 and T47D, were grown as a monolayer in RPMI-1640 plus 10% FCS. H630 and H630-1 cells were grown as previously described (14)(15)(16). Except as noted otherwise, cultured cells were incubated at 37˚C and all media were supplemented with penicillin (100 IU/ ml) and streptomycin (100 μg/ml).…”

Antisense as an explanatory, experimental and therapeutic tool for psychiatric disorders

“…In higher animals, including humans, antisense genes only seem to exist as fragments, and they lack some of the features shared by normal (sense) genes. At least one such antisense gene fragment is expressed in the brain (Adelman et al 1987;Takayama & Inouye, 1990;Dolnick, 1993). In addition, there are genes that have a sequence complementary to a sense gene, or part of it, but which are encoded at a different site in the genome; they, therefore, cannot be called true antisense genes, but their sequence complementarity suggests they have antisense-like effects.…”