A Tumor-Promoting Phorbol Ester Causes a Large Increase in APOBEC3A Expression and a Moderate Increase in APOBEC3B Expression in a Normal Human Keratinocyte Cell Line without Increasing Genomic Uracils

Abstract

Phorbol 12-myristate 13-acetate (PMA) promotes skin cancer in rodents. The mutations found in murine tumors are similar to those found in human skin cancers, and PMA promotes proliferation of human skin cells. PMA treatment of human keratinocytes increases the synthesis of APOBEC3A, an enzyme that converts cytosines in single-stranded DNA to uracil, and mutations in a variety of human cancers are attributed to APOBEC3A or APOBEC3B expression. We tested here the possibility that induction of APOBEC3A by PMA causes genomic accumulation of uracils that may lead to such mutations. When a human keratinocyte cell line was treated with PMA, both APOBEC3A and APOBEC3B gene expression increased, anti-APOBEC3A/APOBEC3B antibody bound a protein(s) in the nucleus, and nuclear extracts displayed cytosine deamination activity. Surprisingly, there was little increase in genomic uracils in PMA-treated wild-type or uracil repair-defective cells. In contrast, cells transfected with a plasmid expressing APOBEC3A acquired more genomic uracils. Unexpectedly, PMA treatment, but not APOBEC3A plasmid transfection, caused a cessation in cell growth. Hence, a reduction in single-stranded DNA at replication forks may explain the inability of PMA-induced APOBEC3A/APOBEC3B to increase genomic uracils. These results suggest that the proinflammatory PMA is unlikely to promote extensive APOBEC3A/APOBEC3B-mediated cytosine deaminations in human keratinocytes.

The APOBEC3 genes and their role in cancer: insights from human papillomavirus

“…Depending on the cell type examined, both A3A and A3B induction has been reported upon activation of PKC signalling: A3A was originally identified as Phorbolin-1, a protein enriched in psoriatic keratinocytes that could be induced by treatment of normal keratinocytes with PMA (Rasmussen & Celis 1993, Madsen et al 1999, while A3B but not A3A, is induced following PMA treatment of the mammary epithelial cell line, MCF10A (Leonard et al 2015). Conversely, while a recent study conducted in normal oral keratinocytes (Siriwardena et al 2018) and our own observations using NIKS in which we have epitope-tagged the endogenous A3A and A3B genes (Smith & Fenton unpublished) confirm a strong, protein kinase C (PKC)dependent increase in A3A protein expression upon PMA treatment, A3B mRNA is induced to a far lesser extent in keratinocytes, with minimal or no detectable increase in A3B protein.…”

“…Human keratinocytes express several TLRs, among which TLR9 is activated by DNA-containing unmethylated CpG motifs including a region from the HPV16 E6 gene (Hasan et al 2007, Lebre et al 2007). In addition to type 1 interferons it induces tumour necrosis factor (TNFα), which has recently been shown to upregulate A3A in keratinocytes (Amcheslavsky et al 2004, Siriwardena et al 2018. The suppression of TLR9 by E7 provides further evidence of its importance in the innate immune responses to HPV (Hasan et al 2007(Hasan et al , 2013.…”

Genome-wide mapping of regions preferentially targeted by the human DNA-cytosine deaminase APOBEC3A using uracil-DNA pulldown and sequencing

“…Although there is no established method for the mapping of uracils in DNA, Bryan et al (29) described two closely related methods to map uracils in DNA that depend on the generation of double-strand breaks at uracils by combining Ung with endonuclease IV, and have used it to map uracils in the HIV-1 genome (30). This method requires that the DNA is highly uracilated (Ͼ1% of cytosines converted to uracil) (29,31) and is not applicable to situations where expression of AID/APOBEC enzymes cause only small increases in genomic uracil levels (a few uracils per 10 6 bp) (32). A different method replaces the uracil with a biotinylated uracil or cytosine using complete BER and uses the biotin tag to pulldown the DNA fragments for sequencing (33) and requires a large number of biochemical steps ( Fig.…”

A rabbit monoclonal antibody against the antiviral and cancer genomic DNA mutating enzyme APOBEC3B

“…The 5210-87-13 mAb has also been shared with colleagues, who have independently demonstrated utility in anti-A3B IB experiments 66,67 . In addition, another independent study used 5210-87-13 to monitor A3A and A3B induction in IB and IF experiments 68 . Thus, depending on the experimental system and biological question, the 5210-87-13 mAb detailed here may be useful for studying human A3A, A3B, and A3G.…”