Visualization of O⁶-methylguanine in target cell nuclei of dimethylnitrosamine-treated human pancreas by a murine monoclonal antibody

Abstract

A murine monoclonal IgG2b with high affinity and specificity for O6-methylguanine was developed. This allowed visualization of this adduct in paraffin-embedded sections by indirect fluorescence or immunoperoxidase methods. The antibody was used to study and compare the formation and persistence of this adduct detected by immunohistochemistry and the presence of 14C methyl by autoradiography in sections from adult human pancreas explants exposed to [14C methyl]dimethylnitrosamine (DMN) and organ-cultured for up to 7 days. O6-methylguanine was detectable randomly and with varied intensity in parenchymal cell nuclei and cytoplasms, 6 h after exposure to DMN. Following RNase treatment, however, only 7.5% of nuclei revealed measurable staining. The positive nuclei decreased to 2.5% within 24 h and remained about the same throughout the 7 days of culture. This correlated well with the change in percentage of positive nuclei for 14C methyl autoradiographic grains which were selectively condensed over the O6-methylguanine-stained nuclei. The data demonstrate a subpopulation of repair-deficient parenchymal cells of human pancreas exposed to DMN.

O6-Methylguanosine leads to position-dependent effects on ribosome speed and fidelity

“…Endogenous and exogenous alkylating agents are known to react with and modify the nitrogen and oxygen atoms of all nucleobases (for review, see Wurtmann and Wolin 2009). RNA appears to be more vulnerable to damage presumably due to its prevalent single-stranded nature exposing the Watson-Crick face of the nucleobases (Parsa et al 1987;Hofer et al 2005). Unlike programmed methylation of specific nucleotides in rRNA and tRNA, aberrant methylation has the potential to deleteriously alter an RNA's function.…”

“…Moreover, organisms from bacteria to man have evolved specific mechanisms to mediate m 6 G lesions in DNA, underscoring the risk of its accumulation (Sedgwick et al 2007). And yet, despite having known for a number of years that m 6 G also accumulates in RNA (Parsa et al 1987), almost nothing is known about its effects on mRNA and specifically how it is decoded by the ribosome.…”