Murray, P. M. | bioRxiv Science

bioRxiv2024

Sensitivity to temozolomide of cell lines derived from brain tumours and melanomas: relationship to MGMT gene expression

BackgroundWe have developed a series of low-passage melanoma and glioma cell lines and are using them to study responses to antitumour drugs. Here we have assessed responses to temozolomide, a commonly used clinical drug used for the treatment of glioblastoma and melanoma. Temozolomide acts mainly by methylation of DNA guanine and lesions can be removed by the repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). Low expression of MGMT is one of the main features associated with sensitivity to temozolomide and is usually associated with CpG methylation of the MGMT promoter. We wished to determine whether temozolomide sensitivity in these lines was related to MGMT promoter methylation and MGMT mRNA synthesis. Methods: 63 melanoma and 37 glioblastoma lines were derived from developed from surgical samples, using atmosphere of 5% oxygen for development and propagation in order to minimise oxygen toxicity. Temozolomide sensitivity (IC50 data) was analysed by its effect on 3H-thymidine incorporation in 5-day assays. MGMT promoter methylation status was assessed by the methylation-specific polymerase chain reaction (PCR). MGMT mRNA expression was assessed using reverse transcription and PCR. Results: 40% of the melanoma lines and 24% of the glioblastoma showed some sensitivity to temozolomide. Of the 79 cell lines analysed for MGMT mRNA expression, a good correlation was found to temozolomide resistance (p < 0.0001). Of the 100 cell lines assessed for MGMT promoter status, 12 showed methylation of both promoter sequences and were all temozolomide-sensitive; 50 lines showed no MGMT promoter methylation and 90% of these were temozolomide resistant; 30 lines showed partial promoter methylation and 53% of these were temozolomide resistant. Conclusion: Temozolomide sensitivity matched MGMT mRNA expression well but only partially matched MGMT promoter methylation. Other factors, such as loss of DNA mismatch repair capacity, may contribute to resistance.

cancer biology↗

bioRxiv2023

HIV, asymptomatic STI, and the rectal mucosal immune environment among young men who have sex with men

Young men who have sex with men (YMSM) are disproportionately affected by HIV and bacterial sexually transmitted infections (STI) including gonorrhea, chlamydia, and syphilis; yet research into the immunologic effects of these infections is typically pursued in siloes. Here, we employed a syndemic approach to understand potential interactions of these infections on the rectal mucosal immune environment among YMSM. We enrolled YMSM aged 18-29 years with and without HIV and/or asymptomatic bacterial STI and collected blood, rectal secretions, and rectal tissue biopsies. YMSM with HIV were on suppressive antiretroviral therapy (ART) with preserved blood CD4 cell counts. We defined 7 innate and 19 adaptive immune cell subsets by flow cytometry, the rectal mucosal transcriptome by RNAseq, and the rectal mucosal microbiome by 16s rRNA sequencing and examined the effects of HIV and STI and their interactions. We measured tissue HIV RNA viral loads among YMSM with HIV and HIV replication in rectal explant challenge experiments among YMSM without HIV. HIV, but not asymptomatic STI, was associated with profound alterations in the cellular composition of the rectal mucosa. We did not detect a difference in the microbiome composition associated with HIV, but asymptomatic bacterial STI was associated with a higher probability of presence of pathogenic taxa. When examining the rectal mucosal transcriptome, there was evidence of statistical interaction; asymptomatic bacterial STI was associated with upregulation of numerous inflammatory genes and enrichment for immune response pathways among YMSM with HIV, but not YMSM without HIV. Asymptomatic bacterial STI was not associated with differences in tissue HIV RNA viral loads or in HIV replication in explant challenge experiments. Our results suggest that asymptomatic bacterial STI may contribute to inflammation particularly among YMSM with HIV, and that future research should examine potential harms and interventions to reduce the health impact of these syndemic infections. AUTHOR SUMMARYYoung men who have sex with men (YMSM) are disproportionately affected by HIV and asymptomatic bacterial sexually transmitted infections (STI) including gonorrhea, chlamydia, and syphilis. However, the health effects of these infections are not typically studied together. In this study, we enrolled YMSM ages 18-29 with and without HIV and/or asymptomatic bacterial STI to study the immunologic effects of these infections, and their interactions, on the rectal mucosa. We found that HIV was associated with differences in the cellular make-up of the rectal tissues, and that STI was associated with an increase in the detection of potentially dangerous bacteria in the rectum. When we examined tissue gene expression, we found that STI was associated with inflammation only among YMSM with HIV, but not those without HIV. We did not see an effect of STI on differences in tissue viral loads among YMSM with HIV or in HIV replication in rectal explant experiments in YMSM without HIV. Our results suggest that asymptomatic bacterial STI may contribute to inflammation particularly among YMSM with HIV, and that future research should examine potential harms and interventions to reduce the health impact of these syndemic infections.

immunology↗