Quantification of ribonucleotide reductase expression in wild-type and hydroxyurea-resistant cell lines employing in situ reverse transcriptase polymerase chain reaction and a computerized image analysis system

Ribonucleotide reductase (RR) is the enzyme responsible for converting nucl eoside diphosphates to deoxynucleoside diphosphates, ensuring a balanced su pply of deoxyribonucleotides for DNA synthesis. Expression of RR is tightly regulated, but it is affected by exogenous agents, such as hydroxyurea (HU ), which inactivates the tyrosyl free radical on the small subunit of RR, R 2. We have previously employed in situ reverse transcriptase (RT)-PCR to es timate expression of R2 in wild-type and HU-resistant human colon carcinoma cell lines and to correlate altered expression of R2 with changes in cell size and morphology. The current studies were undertaken to render this met hodology more quantitative. Both wild-type and resistant cells were grown o n partitioned glass slides and analyzed with in situ RT-PCR. Because both w ildtype and resistant cells were analyzed under a single cover slip, protea se digestion, reverse transcription, PCR, and color development were all pe rformed under identical conditions. Images were analyzed with NIH Image 1.5 9 software. There was a highly significant correlation between expression o f R2 and cell size for both sensitive and resistant cells (P = 0.0001, for both). When cell size was compared either with expression of R2 or cell sha pe, however, these correlated only in wild-type cells (P = 0.001 and 0.0001 , respectively). These data demonstrate that normal cell growth in the unpe rturbed wild-type cell line was closely linked to expression of R2, whereas in the resistant variants which overexpress R2, these correlations were ab sent, suggesting that HU resistance is related to loss of linkage between R 2 expression and cell growth and confirming previous data relating overexpr ession of R2 with multiple other changes in the cell growth repertoire. Thu s, we have demonstrated for the first time a quantitative application of in situ RT-PCR. (C) 1999 Academic Press.