Inducible tumor difference between rapid and slow rat Nat2 congenic Fischer 344 rats administered methyl-nitrosourea.

Human arylamine N-acetyltransferase 1 (NAT1) is a well-known phase II metabolic enzyme that has been associated with carcinogenesis. Its role in the biotransformation of aromatic and heterocyclic amine carcinogens has been investigated for many years, but more recent investigations focus on a possible endogenous role of human NAT1 in cancer initiation and progression. We conducted in vivo studies using homozygous Fischer 344 rats, congenic at the rat Nat2 locus for high (rapid) and low (slow) activity. Wistar Kyoto inbred rats were used to breed in the slow activity rat Nat2 locus into the Fischer 344 inbred rat, which contains the rapid activity rat Nat2 locus. The rat Nat2 gene is a functional ortholog for the human NAT1 because it has similar sequence and substrate specificity to human NAT1. Chemically induced breast tumors are produced in the rat following administration of methyl-nitrosourea (MNU). In this thesis, rapid and slow acetylator female congenic rats were administrated a single dose of MNU (50 mg/kg) by intraperitoneal injection at three weeks of age. Weekly measurements of weights and palpable breast tumors were recorded. Palpable breast tumors showed a significantly shorter latency in rapid compared to slow acetylator congenic rats (p=0.040). At 23 weeks post MNUadministration, rats were euthanized, and tumor and adjacent non-tumor tissue were collected. Tumors were found in 78% of the rapid acetylator congenic rats with an average ± SEM of 1.78 ± 0.7 tumors per rat. In contrast, tumors were found in only 30% of slow acetylator congenic rats with an average of 0.5 ± 0.3 tumors per rat. Both tumor multiplicity and incidence approached significance (p=0.073 and 0.069, respectively) in this initial pilot experiment. Histopathology of the tumors classified the majority of the tumors as intraductal papillomas that were estrogen receptor positive by immunohistochemistry. The miRNA 574-3p was under expressed in intraductal papilloma breast tumors compared to normal tissues. These results suggest an important role for rat Nat2 in MNU-induced carcinogenesis and possibly carcinogenesis in general. Additional studies are proposed to confirm and understand the mechanism of rat Nat2’s involvement in carcinogenesis

Congenic rats with higher arylamine N-acetyltransferase 2 activity exhibit greater carcinogen-induced mammary tumor susceptibility independent of carcinogen metabolism

Abstract Background Recent investigations suggest role(s) of human arylamine N-acetyltransferase 1 (NAT1) in breast cancer. Rat NAT2 is orthologous to human NAT1 and the gene products are functional homologs. We conducted in vivo studies using F344.WKY-Nat2 rapid/slow rats, congenic at rat Nat2 for high (rapid) and low (slow) arylamine N-acetyltransferase activity, to assess a possible role for rat NAT2 in mammary tumor susceptibility. Methods Mammary carcinogens, methylnitrosourea (MNU) and 7,12-dimethylbenzanthracene (DMBA) neither of which is metabolized by N-acetyltransferase, were administered to assess mammary tumors. MNU was administered at 3 or 8 weeks of age. DMBA was administered at 8 weeks of age. NAT2 enzymatic activity and endogenous acetyl-coenzyme A (AcCoA) levels were measured in tissue samples and embryonic fibroblasts isolated from the congenic rats. Results Tumor latency was shorter in rapid NAT2 rats compared to slow NAT2 rats, with statistical significance for MNU administered at 3 and 8 weeks of age (p = 0.009 and 0.050, respectively). Tumor multiplicity and incidence were higher in rapid NAT2 rats compared to slow NAT2 rats administered MNU or DMBA at 8 weeks of age (MNU, p = 0.050 and 0.035; DMBA, p = 0.004 and 0.027, respectively). Recombinant rat rapid-NAT2, as well as tissue samples and embryonic fibroblasts derived from rapid NAT2 rats, catalyzed p-aminobenzoic acid N-acetyl transfer and folate-dependent acetyl-coenzyme A (AcCoA) hydrolysis at higher rates than those derived from rat slow-NAT2. Embryonic fibroblasts isolated from rapid NAT2 rats displayed lower levels of cellular AcCoA than slow NAT2 rats (p < 0.01). Conclusions A novel role for rat NAT2 in mammary cancer was discovered unrelated to carcinogen metabolism, suggesting a role for human NAT1 in breast cancer

Proteomic analysis of arylamine N-acetyltransferase 1 knockout breast cancer cells: Implications in immune evasion and mitochondrial biogenesis

Previous studies have shown that inhibition or depletion of N-acetyltransferase 1 (NAT1) in breast cancer cell lines leads to growth retardation both in vitro and in vivo, suggesting that NAT1 contributes to rapid growth of breast cancer cells. To understand molecular and cellular processes that NAT1 contributes to and generate novel hypotheses in regard to NAT1′s role in breast cancer, we performed an unbiased analysis of proteomes of parental MDA-MB-231 breast cancer cells and two separate NAT1 knockout (KO) cell lines. Among 4890 proteins identified, 737 proteins were found significantly (p < 0.01) upregulated, and 651 proteins were significantly (p < 0.01) downregulated in both NAT1 KO cell lines. We performed enrichment analyses to identify Gene Ontology biological processes, molecular functions, and cellular components that were enriched in each data set. Among the proteins upregulated in NAT1 KO cells, pathways associated with MHC (major histocompatibility complex) I-mediated antigen presentation were significantly enriched. This raises an interesting and new hypothesis that upregulation of NAT1 in breast cancer cells may aid them evade immune detection. Multiple pathways involved in mitochondrial functions were collectively downregulated in NAT1 KO cells, including multiple subunits of mitochondrial ATP synthase (Complex V of the electron transport chain). This was accompanied by a reduction in cell cycle-associated proteins and an increase in pro-apoptotic pathways in NAT1 KO cells, consistent with reported observations that NAT1 KO cells exhibit a slower growth rate both in vitro and in vivo. Thus, mitochondrial dysfunction in NAT1 KO cells likely contributes to growth retardation