Cellular Responses to Anthracyclines Identify Ku70, a DNA Repair Factor that Changes Compartment and Remains Stable in Leukemic Cells

Anthracyclines such as doxorubicin and daunorubicin are anticancer drugs that act by damaging the DNA and used for treating a variety of cancers including adult acute myeloid leukemia.  To date, nearly 50 % of acute myeloid leukemia patients show resistance to anthracyclines although the cause is not known.  We first investigate if there is a relationship between the expression level of 23 DNA repair genes in three leukemic cell lines (KG-1, HL-60 and Mono-Mac1) and cellular responses to anthracyclines.  We observed that the DNA repair genes were all downregulated in these cell lines following exposure to doxorubicin.  Further analysis revealed that the general downregulation of the genes was linked to a substantial decrease in the recovery of total RNA raising the possibility that assessment of total RNA, and not specific gene or set of genes, can be used as a simple indicator of cellular responses to anthracyclines.  Furthermore, examination of total protein extracts derived from these cell lines revealed for the first time that Ku70 is a key protein that remained stable, while the majority of proteins were loss, upon anthracycline treatment.  Importantly, Ku70 redistributes from the cytoplasm to the nucleoli in a time-dependent manner in response to anthracycline exposure.  We propose that Ku70 redistribution might play a vital role in predicting cellular response to anthracycline and promoting cell death

The Peptidyl Prolyl Isomerase Rrd1 Regulates the Elongation of RNA Polymerase II during Transcriptional Stresses

Rapamycin is an anticancer agent and immunosuppressant that acts by inhibiting the TOR signaling pathway. In yeast, rapamycin mediates a profound transcriptional response for which the RRD1 gene is required. To further investigate this connection, we performed genome-wide location analysis of RNA polymerase II (RNAPII) and Rrd1 in response to rapamycin and found that Rrd1 colocalizes with RNAPII on actively transcribed genes and that both are recruited to rapamycin responsive genes. Strikingly, when Rrd1 is lacking, RNAPII remains inappropriately associated to ribosomal genes and fails to be recruited to rapamycin responsive genes. This occurs independently of TATA box binding protein recruitment but involves the modulation of the phosphorylation status of RNAPII CTD by Rrd1. Further, we demonstrate that Rrd1 is also involved in various other transcriptional stress responses besides rapamycin. We propose that Rrd1 is a novel transcription elongation factor that fine-tunes the transcriptional stress response of RNAPII

Modulation of Gene Expression in Human Macrophages Treated with the Anti-Leishmania Pentavalent Antimonial Drug Sodium Stibogluconate▿ †

Within the mammalian host, Leishmania donovani is an obligatory intracellular protozoan parasite that resides and multiplies exclusively in the phagolysosomes of macrophages. Leishmania control relies primarily on chemotherapy, with the mainstay being pentavalent antimony (SbV) complexed to carbohydrates in the form of sodium stibogluconate (Pentostam) or meglumine antimoniate (Glucantime). The mode of action of SbV is still not known precisely. To explore the effect of SbV on macrophage gene expression, a microarray analysis was performed using Affymetrix focus arrays to compare gene expression profiles in noninfected and L. donovani-infected THP-1 monocytic cells treated or not treated with sodium stibogluconate. Under our experimental conditions, SbV changed the expression of a few host genes, and this was independent of whether cells were infected or not infected with Leishmania. Leishmania infection had a greater effect on the modulation of host gene expression. Statistical analyses have indicated that the expression of eight genes was modified by at least twofold upon SbV treatment, with six genes upregulated and two genes downregulated. One gene whose expression was affected by SbV was the heme oxygenase gene HMOX-1, and this change was observed both in the monocytic cell line THP-1 and in primary human monocyte-derived macrophages. Another pathway that was affected was the glutathione biosynthesis pathway, where the expression of the glutamate-cysteine ligase modifier subunit was increased upon SbV treatment. Our analysis has suggested that, under our experimental conditions, the expression of a few genes is altered upon SbV treatment, and some of these encoded proteins may be implicated in the yet-to-be-defined mode of action of SbV

Role of the ABC Transporter MRPA (PGPA) in Antimony Resistance in Leishmania infantum Axenic and Intracellular Amastigotes

Antimonial compounds are the mainstay for the treatment of infections with the protozoan parasite Leishmania. We present our studies on Leishmania infantum amastigote parasites selected for resistance to potassium antimonyl tartrate [Sb(III)]. Inside macrophages, the Sb(III)-selected cells are cross-resistant to sodium stibogluconate (Pentostam), the main drug used against Leishmania. Putative alterations in the level of expression of more than 40 genes were compared between susceptible and resistant axenic amastigotes using customized DNA microarrays. The expression of three genes coding for the ABC transporter MRPA (PGPA), S-adenosylhomocysteine hydrolase, and folylpolyglutamate synthase was found to be consistently increased. The levels of cysteine were found to be increased in the mutant. Transfection of the MRPA gene was shown to confer sodium stibogluconate resistance in intracellular parasites. This MRPA-mediated resistance could be reverted by using the glutathione biosynthesis-specific inhibitor buthionine sulfoximine. These results highlight for the first time the role of MRPA in antimony resistance in the amastigote stage of the parasite and suggest a strategy for reversing resistance