Antonio T. Baines

Lytic transglycosylase A in Neisseria gonorrhoeae cleaves the β-1,4-glycosidic bond between peptidoglycan (PG) monomers to liberate 1,6-anhydro-PG fragments that are either recycled or released as cytotoxic fragments. To gain further insight into the effect of LtgA on cellular processes in Neisseria gonorrhoeae, we performed a proteomic analysis comparing wild-type and an isogenic ltgA null mutant strain. Proteins were separated by two-dimensional gel electrophoresis and identified by MALDI-TOF mass spectrometry, which revealed several proteins that were increased in their level of expression upon loss of LtgA. The most notable changes corresponded to enzymes related to aminosugar and pyrimidine metabolism. Quantitative real-time RT-PCR of mRNA from a ltgA null strain confirmed increased transcription of genes encoding enzymes involved in UDP-N-acetylglucosamine (UDP-GlcNAc) synthesis, a major precursor in PG and lipooligosaccharide (LOS) synthesis, during normal growth conditions and following exposure to penicillin. We also found that the ltgA mutant strains were more susceptible to β-lactam antibiotics, vancomycin, and the human-cathelicidin antibacterial peptide, LL-37, than their corresponding wild-type parental strains. Our results suggest that increased expression of enzymes responsible for production UDP-GlcNAc is an adaptive response due to inactivation of ltgA and/or exposure to penicillin.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with a 5-year survival rate of only 6%. Although the cytosine analog gemcitabine is the drug commonly used to treat PDAC, chemoresistance unfortunately renders the drug ineffective. Thus, strategies that can decrease this resistance will be essential for improving the dismal outcome of patients suffering from this disease. We previously observed that oncogenic Pim-1 kinase was aberrantly expressed in PDAC tissues and cell lines and was responsible for radioresistance. Furthermore, members of the Pim family have been shown to reduce the efficacy of chemotherapeutic drugs in cancer. Therefore, we attempted to evaluate the role of Pim-3 in chemoresistance of PDAC cells. We were able to confirm upregulation of the Pim-3 oncogene in PDAC tissues and cell lines vs. normal samples. Biological consequences of inhibiting Pim-3 expression with shRNA-mediated suppression included decreases in anchorage-dependent growth, invasion through Matrigel and chemoresistance to gemcitabine as measured by caspase-3 activity. Additionally, we were able to demonstrate that Pim-1 and Pim-3 play overlapping but non-identical roles as it relates to gemcitabine sensitivity of pancreatic cancer cells. To further support the role of Pim-3 suppression in sensitizing PDAC cells to gemcitabine, we used the pharmacological Pim kinase inhibitor SGI-1776. Treatment of PDAC cells with SGI-1776 resulted in decreased phosphorylation of the proapoptotic protein Bad and cell cycle changes. When SGI-1776 was combined with gemcitabine, there was a greater decrease in cell viability in the PDAC cells vs. cells treated with either of the drugs separately. These results suggest combining drug therapies that inhibit Pim kinases, such as Pim-3, with chemotherapeutic agents, to aid in decreasing chemoresistance in pancreatic cancer.

The RAS oncogenes (HRAS, NRAS and KRAS) comprise the most frequently mutated class of oncogenes in human cancers (33%), thus stimulating intensive effort in developing anti-Ras inhibitors for cance...

Oncogenic Pim-1 kinase is upregulated in multiple solid cancers, including human pancreatic ductal adenocarcinoma (PDAC), a highly lethal disease with few useful treatment options. Pim-1 is also transcriptionally induced upon oncogenic K-Ras-mediated transformation of the human pancreatic ductal epithelial (HPDE) cell model of PDAC. Given the near ubiquitous presence of mutant K-Ras in PDAC and its critical role in this disease, we wished to study the effects of oncogenic K-Ras signaling on Pim-1 expression, as well as the role of Pim-1 in growth transformation of PDAC cells. Pim-1 protein levels were upregulated in both PDAC cell lines and patient tumor tissues. Furthermore, ectopic oncogenic K-Ras increased Pim-1 expression in human pancreatic nestin-expressing (HPNE) cells, a distinct immortalized cell model of PDAC. Conversely, shRNA-mediated suppression of oncogenic K-Ras decreased Pim-1 protein in PDAC cell lines. These results indicate that oncogenic K-Ras regulates Pim-1 expression. The kinase activity of Pim-1 is constitutively active. Accordingly, shRNA-mediated suppression of Pim-1 in K-Ras-dependent PDAC cell lines decreased Pim-1 activity, as measured by decreased phosphorylation of the pro-apoptotic protein Bad and increased expression of the cyclin-dependent kinase inhibitor p27 ^Kip1 . Biological consequences of inhibiting Pim-1 expression included decreases in both anchorage-dependent and -independent cell growth, invasion through Matrigel and radioresistance as measured by standard clonogenic assays. These results indicate that Pim-1 is required for PDAC cell growth, invasion and radioresistance downstream of oncogenic K-Ras. Overall, our studies help to elucidate the role of Pim-1 in PDAC growth transformation and validate Pim-1 kinase as a potential molecular marker for mutated K-Ras activity.

(1995). Alterations in Chemically Induced Tissue Injury Related to All-trans-Retinol Pretreatment In Rodents. Drug Metabolism Reviews: Vol. 27, No. 1-2, pp. 299-323.