We previously demonstrated that pentamidine, which has been clinically used against Pneumocystis carinii, inhibits in vitro a group I intron ribozyme from that organism. Another fungal pathogen, Candida albicans, also harbors a group I intron ribozyme (Ca.LSU) in the essential rRNA genes in almost half of the clinical isolates analyzed. To determine whether pentamidine inhibits Ca.LSU in vitro and in cells, phylogenetically closely related intron-containing (4-1) and intronless (62-1) strains were studied. Splicing in vitro of the Ca.LSU group I intron ribozyme was completely inhibited by pentamidine at 200 M. On rich glucose medium, the intron-containing strain was more sensitive to growth inhibition by pentamidine than was the intronless strain, as measured by disk or broth microdilution assays. On rich glycerol medium, they were equally susceptible to pentamidine. At pentamidine levels selectively inhibiting the intron-containing strain (1 M) in glucose liquid cultures, inhibition of splicing and rRNA maturation was detected by quantitative reverse transcription-PCR within 1 min with a 10-to 15-fold accumulation of precursor rRNA. No comparable effect was seen in the intronless strain. These results correlate the cellular splicing inhibition of Ca.LSU with the growth inhibition of strain 4-1 harboring Ca.LSU. Broth microdilution assays of 13 Candida strains showed that introncontaining strains were generally more susceptible to pentamidine than the intronless strains. Our data suggest that ribozymes found in pathogenic microorganisms but absent in mammals may be targets for antimicrobial therapy.

Creation of large (∼15 kb) recombinant plasmids can be done in a single step by co‐transformation of yeast cells with a partial restriction digest of a plasmid vector and a linear insert whose ends overlap one of the vector restriction sites. This method is used to generate a plasmid expressing the Saccharomyces cerevisiae rRNA genes containing the Ca.LSU group I intron ribozyme from Candida albicans. This plasmid expresses functional rRNA and ribozyme. Copyright © 2000 John Wiley & Sons, Ltd.

The main causes of treatment failure and mortality in cancer are metastases and the development of drug resistance. CD44 and P-gp are two membrane proteins well-known determinants of metastases and drug resistance respectively. We and others have shown that CD44 induces P-gp expression in cancer cells. We also showed that there is a physical interaction between these two proteins as they immunoprecipitate and co-localize in the cell membrane. Hyaluronan (HA) is a well known ligand for CD44, involved in tumor cell signaling and the development of malignant properties. Others have shown that HA is necessary for the CD44 induction of P-gp expression and drug resistance. However, it is not known whether HA binding to CD44 is indeed necessary for this induction. To answer this question, we generated different mutants of the CD44 HA binding domains (CD44 HABD). We transfected CD44 HABD mutants as well as CD44 wt into ovarian cancer cells (TOV112D) and human embryonic kidney cell lines (HEK 293). Unexpectedly, we found that P-gp expression was induced both in CD44 wt and CD44 HABD mutants. However, the P-gp induction was significantly less in the CD44 HABD mutants. We tested whether the P-gp induced by the CD44-HABD mutants was functional by testing for drug sensitivity in an MTT assay. We observed that cells transfected with CD44 HABD mutants became drug sensitive as compared to CD44 wt transfected cells even when the whole HA binding domain was deleted. We then determined if deletion of the CD44 HABD domain had an effect on the physical interaction between CD44 and P-gp. By co-immunoprecipitation experiments we showed that CD44 and P-gp were physically interacting even in CD44 HABD mutants. These results indicate the existence of an additional mechanism for P-gp induction through CD44 that is independent of HA binding. Previously, we showed that the intracytoplasmic domain of CD44 (CD44-ICD) is transported into the nucleus where it binds to DNA promoters and is involved in the transcriptional regulation of various genes. Therefore, we investigated whether CD44 was involved in transcriptional regulation of P-gp as the additional mechanism of Pgp upregulation. Co-transfection of CD44 or CD44-HABD mutants with luciferase driven MDR1 promoter showed increased luciferase activity in both CD44wt and CD44-HABD mutants. However, this MDR1 promoter does not have the CD44 DNA binding consensus sequence. Therefore, CD44-ICD could be inducing other genes that in turn activate the MDR1 gene. We conclude that although HA binding to CD44 is important for the induction of P-gp, CD44 transcriptional activation of MDR1 promoter also plays a role. Furthermore, we show that CD44 transcriptional activation of MDR1 is independent of HA binding. These results further the understanding to the present knowledge of CD44 involvement in drug resistance and uncovers new mechanisms involved in this process that are HA-independent. Citation Format: Swayamjot Kaur, Kyle Murphy, Karl Miletti, Abhilash Ravindranath, Lorna Rodriguez-Rodriguez. CD44 induces P-gp expression through hyaluronic acid binding and transcriptional activation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 758. doi:10.1158/1538-7445.AM2014-758