Natalia Ignatenko

Interests

Research

For the past 20 years I have been involved in colorectal cancer (CRC) research with the focus on the molecular mechanisms of detection and prevention. My research activity include identification and validation of downstream effectors of Adenomatous Polyposis Coli (APC) tumor suppressor gene and K-ras protooncogene within polyamine and prostaglandin pathways and study the luminal and dietary risk factors of colonic inflammation and colorectal carcinogenesis. I also have an expertise in developing Genetically Engineered Mouse (GEM) models of gastrointestinal cancer for studying different steps of carcinogenesis.Current research in my laboratory is focused on regulation and pathophysiological functions of kallikrein-related peptidase 6 (KLK6) in cancer with the focus on cancer cells invasion and metastasis. KLK6 is a secreted serine protease with the specific function to cleave components of the extracellular matrix and activate latent growth factors involved in epithelial-to-mesenchymal transition (EMT) pathways. Our recent findings indicate that KLK6 expression in colon cancer cells is determined by the tumor subtype and the genetic mutations developed during the colon cancer progression. KLK6 protein will be useful for imaging purposes and an appropriate target for evaluation as a colon biomarker.

Teaching

Although I am not currently involved in formal teaching, I would be interested in participation in formal academic curriculum to enrich students' training with my research experiences and broad background in cancer biology . The possible themes are: biochemistry and molecular biology of polyamines in colon carcinogenesis, the molecular mechanisms of gastrointestinal malignancies and development of novel strategies for prevention, detection and treatment of these malignancies.

Courses

Scholarly Contributions

Chapters

• Gerner, E. W., Ignatenko, N. A., & Fultz, K. E. (2002). Inducible gene expression strategies in gene therapy. In Pharmaceutical perspectives of nucleic acid-based therapeutics(pp 15-33). Taylor & Francis. doi:10.4324/9780203300961_CHAPTER_2

Journals/Publications

• Georgieva, T., Darmoul, D., Chen, H., Cui, H., Rice, P., Barton, J., Besselsen, D., & Ignatenko, N. (2024). Kallikrein-Related Peptidase 6 Contributes to Murine Intestinal Tumorigenesis Driven by a Mutant Adenomatous polyposis coli Gene. Cancers, 16(22). doi:10.3390/cancers16223842
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  Background/Objectives: The objective of this study was to assess the role of a secreted serine protease, kallikrein-related peptidase 6 (KLK6), during colorectal tumorigenesis driven by a mutant Adenomatous polyposis coli (APC) tumor suppressor gene. A first analysis of KLK6 expression in the intestinal tract of Apc-mutant multiple intestinal neoplasia (ApcMin/+) mice revealed up to four-fold induction of Klk6 mRNA levels in adenomas relative to its level in the adjacent mucosa. Methods and Results: The presence of KLK6 protein in the adenomatous areas was confirmed by immunohistochemistry and optical coherence tomography/laser-induced fluorescence (OCT/LIF) imaging. To assess the contribution of the KLK6 expression on the Apc-mutant intestinal and colon tumorigenesis, we engineered a mouse with floxed alleles of the Klk6 gene (Klk6lox/lox) and crossed it with a mouse expressing the truncated APC protein under control of the intestinal tract-specific human CDX2P9.5-NLS Cre transgene (CPC;Apcfl/fl;Klk6+/+). We found that CPC;Apcfl/fl mice with disrupted Klk6 gene expression (CPC;Apcfl/fl;Klk6fl/fl) had a significantly smaller average size of the small intestinal and colon crypts (p < 0.001 and p = 0.04, respectively) and developed a significantly fewer adenomas (p = 0.01). Moreover, a decrease in high-grade adenomas (p = 0.03) and adenomas with a diameter above 2 mm (p < 0.0001) was noted in CPC;Apcfl/fl;Klk6fl/fl mice. Further molecular analysis showed that Klk6 gene inactivation in the small intestine and colon tissues of CPC;Apcfl/fl;Klk6fl/fl mice resulted in a significant suppression of transforming growth factor β2 (TGF-β2) protein (p ≤ 0.02) and mitogen-activated protein kinase (MAPK) phosphorylation (p ≤ 0.01). Conclusions: These findings demonstrate the oncogenic role of KLK6 in the mutant Apc-mediated intestinal tumorigenesis and suggest the utility of KLK6 for early diagnosis of colorectal tumors.
• Maar, K. D., Ignatenko, N., Warfel, N. A., Batai, K., Cress, A. E., Pollock, G., Wong, A. C., & Lee, B. R. (2023). Digital Image analysis using Video microscopy of human-derived prostate cancer vs normaprostate organoids to assess migratory behavior on extracellular matrix proteins.. Frontiers in Oncology.
• Bouzid, H., Soualmia, F., Oikonomopoulou, K., Soosaipillai, A., Walker, F., Louati, K., Lo Dico, R., Pocard, M., El Amri, C., Ignatenko, N. A., & Darmoul, D. (2022). Kallikrein-Related Peptidase 6 (KLK6) as a Contributor toward an Aggressive Cancer Cell Phenotype: A Potential Role in Colon Cancer Peritoneal Metastasis. Biomolecules, 12(7).
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  Kallikrein-related peptidases (KLKs) are implicated in many cancer-related processes. KLK6, one of the 15 KLK family members, is a promising biomarker for diagnosis of many cancers and has been associated with poor prognosis of colorectal cancer (CRC) patients. Herein, we evaluated the expression and cellular functions of KLK6 in colon cancer-derived cell lines and in clinical samples from CRC patients. We showed that, although many KLKs transcripts are upregulated in colon cancer-derived cell lines, KLK6, KLK10, and KLK11 are the most highly secreted proteins. KLK6 induced calcium flux in HT29 cells by activation and internalization of protease-activated receptor 2 (PAR2). Furthermore, KLK6 induced extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation. KLK6 suppression in HCT-116 colon cancer cells decreased the colony formation, increased cell adhesion to extracellular matrix proteins, and reduced spheroid formation and compaction. Immunohistochemistry (IHC) analysis demonstrated ectopic expression of KLK6 in human colon adenocarcinomas but not in normal epithelia. Importantly, high levels of KLK6 protein were detected in the ascites of CRC patients with peritoneal metastasis, but not in benign ascites. These data indicate that KLK6 overexpression is associated with aggressive CRC, and may be applied to differentiate between benign and malignant ascites.
• Chen, Y., Darmoul, D., Ignatenko, N. A., Kisiel, C. C., Nfonsam, V., Pandey, R., & Zhou, M. (2021). Molecular pathways associated with Kallikrein 6 overexpression in colorectal cancer. Genes, 749. doi:10.1101/2020.12.09.20245571
• De Vita, E., Smits, N., van den Hurk, H., Beck, E., Hewitt, J., Baillie, G., Russell, E., Pannifer, A., Hamon, V., Morrison, A., McElroy, S., Jones, P., Ignatenko, N., Gunkel, N., & Miller, A. (2020). Synthesis and Structure-Activity Relationships of N-(4-Benzamidino)-Oxazolidinones: Potent and Selective Inhibitors of Kallikrein-Related Peptidase 6. ChemMedChem, 15(1). doi:10.1002/cmdc.201900536
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  Kallikrein-related peptidase 6 (KLK6) is a secreted serine protease that belongs to the family of tissue kallikreins. Aberrant expression of KLK6 has been found in different cancers and neurodegenerative diseases, and KLK6 is currently studied as a potential target in these pathologies. We report a novel series of KLK6 inhibitors discovered in a high-throughput screen within the European Lead Factory program. Structure-guided design based on docking studies enabled rapid progression of a hit cluster to inhibitors with improved potency, selectivity and pharmacokinetic properties. In particular, inhibitors 32 ((5R)-3-(4-carbamimidoylphenyl)-N-((S)-1-(naphthalen-1-yl)propyl)-2-oxooxazolidine-5-carboxamide) and 34 ((5R)-3-(6-carbamimidoylpyridin-3-yl)-N-((1S)-1-(naphthalen-1-yl)propyl)-2-oxooxazolidine-5-carboxamide) have single-digit nanomolar potency against KLK6, with over 25-fold and 100-fold selectivities against the closely related enzyme trypsin, respectively. The most potent compound, 32, effectively reduces KLK6-dependent invasion of HCT116 cells. The high potency in combination with good solubility and low clearance of 32 make it a good chemical probe for KLK6 target validation in vitro and potentially in vivo.
• Chen, H., Henkhaus, R. S., Sells, E., Pandey, R., Abril, E. R., Hsu, C., Krouse, R. R., Nagle, R. B., Pampalakis, G., Sotiropoupou, G., & Ignatenko, N. A. (2019). Kallikrein 6 protease advances colon tumorigenesis via induction of the high mobility group A2 protein. Oncotarget, 10(58), 6062-6068.
• De Vita, E., Smits, N., van den Hurk, H., Beck, E. M., Hewitt, J., Baillie, G., Russell, E., Pannifer, A., Hamon, V., Morrison, A., McElroy, S., Jones, P., Ignatenko, N. A., Gunkel, N., & Miller, A. (2019). Synthesis and structure–activity relationships of N-(4-benzamidino)- oxazolidinones–potent and selective inhibitors of kallikrein-related peptidase 6. ChemMedChem., Epub2019.
• Rice, P. F., Ehrichs, K. G., Jones, M. S., Chen, H., Hsu, C. H., Abril, E. R., Nagle, R. B., Besselsen, D. G., Barton, J. K., & Ignatenko, N. A. (2018). Does Mutated Oncogene Attenuate the Effect of Sulindac in Colon Cancer Chemoprevention?. Cancer prevention research (Philadelphia, Pa.), 11(1), 16-26.
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  The NSAID sulindac has been successfully used alone or in combination with other agents to suppress colon tumorigenesis in patients with genetic predisposition and also showed its efficacy in prevention of sporadic colon adenomas. At the same time, some experimental and clinical reports suggest that a mutant oncogene may negate sulindac antitumor efficacy. To directly assess sulindac activity at suppressing premalignant lesions carrying K-RAS mutation, we utilized a novel mouse model with an inducible colon-specific expression of the mutant oncogene ( ). Tumor development and treatment effects were monitored by minimally invasive endoscopic Optical coherence tomography. Expression of the mutant allele accelerated azoxymethane (AOM)-induced colon carcinogenesis in C57BL/6 mice, a strain otherwise resistant to this carcinogen. Sulindac completely prevented AOM-induced tumor formation in wild-type ( wt) animals. In -mutant mice, a 38% reduction in tumor number, an 83% reduction in tumor volume ( ≤ 0.01) and an increase in the number of adenoma-free mice ( = 0.04) were observed. The partial response of animals to sulindac treatment was evident by the decrease in mucosal thickness ( < 0.01) and delay in progression of the precancerous aberrant crypt foci to adenomas. Molecular analyses showed significant induction in cyclooxygenase 2 (COX-2), cleaved caspase-3 (CC3), and Ki-67 expression by AOM, but not sulindac treatment, in all genotypes. Our data underscore the importance of screening for mutations in individuals with colon polyps to provide more personalized interventions targeting mutant signaling pathways. .
• Sinharay, S., Randtke, E. A., Howison, C. M., Ignatenko, N. A., & Pagel, M. D. (2018). Detection of enzyme activity and inhibition during studies in solution, in vitro, and in vivo with catalyCEST MRI. Molec. Imaging Biol, 20, 240-248.
• Chen, H., Cui, H., Doetschman, T., Ignatenko, N. A., Pampalakis, G., Pandey, R., Sells, E., & Sotiropoulou, G. (2017). Abstract 854: Human tissue Kallikrein 6 enzyme activity regulates epithelial-mesenchymal transition in colon cancer. Cancer Research, 77(13_Supplement), 854-854. doi:10.1158/1538-7445.am2017-854
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  Abstract Background: Kallikrein-related peptidase 6 (KLK6) belongs to the family of human tissue kallikrein genes, majority of which are shown to be differentially expressed in cancers. Clinical studies have demonstrated that upregulation of KLK6 in primary colorectal tumors and lymph nodes correlates with serosal invasion, liver metastasis and indicative of poor prognosis in patients. It has been reported that KLK6 protein is involved in regulation of the epithelial-mesenchymal transition (EMT) program in an organ-specific context. The aim in this study was to investigate contribution of KLK6 enzyme in the EMT during neoplastic transformation in the colon. Results: We expressed enzymatically active or inactive KLK6, using pcDNA3.1(+)preproKLK6 and pcDNA3.1(+)preproKLK6 Ser197Ala mutant plasmids, in Caco-2 colon cancer cell line, which has been characterized before as a very low KLK6 expresser with an undetectable secreted KLK6. Stable isogenic clones were selected and further evaluated for their ability to migrate and invade using in vitro assays and to metastasize in vivo using SCID orthotopic mouse model. We found no effect of KLK6 enzyme activity on migration of Caco-2 cells, expressing the empty vector (Caco-2 mock), and Caco-2 cells, expressing an enzymatically active KLK6 (Caco-2 KLK6 wt) or inactive KLK6 (Caco-2 KLK6 mut). But Caco-2 KLK6 wt cells demonstrated the invasive phenotype in Matrigel invasion assays (p
• Ignatenko, N. A., Cui, H., Skovan, B. A., Chen, H., Pandey, R., & Sells, E. (2017). Specific microRNA-mRNA regulatory network of colon cancer invasion regulated by tissue kallikrein-related peptidase 6. Neoplasia.
• Finnberg, N., Ignatenko, N. A., Lee, N. P., Stairs, D. B., & Yee, N. S. (2015). Animal Models of Cancer Biology. Cancer growth and metastasis, 8s1, CGM.S37907. doi:10.4137/cgm.s37907
• Gerner, E. W., Cremolini, C., Henkhaus, R. S., Ignatenko, N. A., Loupakis, F., & Roy, U. K. (2015). Erratum.. International journal of cancer, 137(8), E18. doi:10.1002/ijc.29624
• LeGendre-McGhee, S., Rice, P. S., Wall, R. A., Sprute, K. J., Bommireddy, R., Luttman, A. M., Nagle, R. B., Abril, E. R., Farrell, K., Hsu, C. H., Roe, D. J., Gerner, E. W., Ignatenko, N. A., & Barton, J. K. (2015). Time-serial Assessment of Drug Combination Interventions in a Mouse Model of Colorectal Carcinogenesis Using Optical Coherence Tomography. Cancer growth and metastasis, 8(Suppl 1), 63-80.
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  Optical coherence tomography (OCT) is a high-resolution, nondestructive imaging modality that enables time-serial assessment of adenoma development in the mouse model of colorectal cancer. In this study, OCT was utilized to evaluate the effectiveness of interventions with the experimental antitumor agent α-difluoromethylornithine (DFMO) and a nonsteroidal anti-inflammatory drug sulindac during early [chemoprevention (CP)] and late stages [chemotherapy (CT)] of colon tumorigenesis. Biological endpoints for drug interventions included OCT-generated tumor number and tumor burden. Immunochistochemistry was used to evaluate biochemical endpoints [Ki-67, cleaved caspase-3, cyclooxygenase (COX)-2, β-catenin]. K-Ras codon 12 mutations were studied with polymerase chain reaction-based technique. We demonstrated that OCT imaging significantly correlated with histological analysis of both tumor number and tumor burden for all experimental groups (P < 0.0001), but allows more accurate and full characterization of tumor number and burden growth rate because of its time-serial, nondestructive nature. DFMO alone or in combination with sulindac suppressed both the tumor number and tumor burden growth rate in the CP setting because of DFMO-mediated decrease in cell proliferation (Ki-67, P < 0.001) and K-RAS mutations frequency (P = 0.04). In the CT setting, sulindac alone and DFMO/sulindac combination were effective in reducing tumor number, but not tumor burden growth rate. A decrease in COX-2 staining in DFMO/sulindac CT groups (COX-2, P < 0.01) confirmed the treatment effect. Use of nondestructive OCT enabled repeated, quantitative evaluation of tumor number and burden, allowing changes in these parameters to be measured during CP and as a result of CT. In conclusion, OCT is a robust minimally invasive method for monitoring colorectal cancer disease and effectiveness of therapies in mouse models.
• LeGendre-McGhee, S., Rice, P. S., Wall, R. A., Sprute, K. J., Bommireddy, R., Luttman, A. M., Nagle, R. B., Abril, E. R., Farrell, K., Hsu, C. H., Roe, D. J., Gerner, E. W., Ignatenko, N. A., & Barton, J. K. (2015). Time-serial Assessment of Drug Combination Interventions in a Mouse Model of Colorectal Carcinogenesis Using Optical Coherence Tomography. Cancer growth and metastasis, 8, 63-80.
• Padavano, J., Henkhaus, R. S., Chen, H., Skovan, B. A., Cui, H., & Ignatenko, N. A. (2015). Mutant K-RAS Promotes Invasion and Metastasis in Pancreatic Cancer Through GTPase Signaling Pathways. Cancer growth and metastasis, 8, 95-113.
• Roy, U., Henkhaus, R., Loupakis, F., Cremolini, C., Gerner, E., & Ignatenko, N. (2015). Erratum: Caveolin-1 is a novel regulator of K-RASdependent migration in colon carcinogenesis (International Journal of Cancer (2013) 133: 1 (43-57) DOI: 10.1002/ijc.28001). International Journal of Cancer, 137(8). doi:10.1002/ijc.29624
• Yee, N. S., Ignatenko, N., Finnberg, N., Lee, N., & Stairs, D. (2015). ANIMAL MODELS OF CANCER BIOLOGY. Cancer growth and metastasis, 8(Suppl 1), 115-8.
• Buckmeier, J. A., Cui, H., Ignatenko, N. A., Krouse, R. S., Nagle, R. B., Pandey, R., Skovan, B. A., & Thompson, P. A. (2014). Abstract 2009: Role of kallikrein 6 secretion in metastatic colon cancer. Cancer Research, 74(19_Supplement), 2009-2009. doi:10.1158/1538-7445.am2014-2009
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  Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Growing evidence indicates that serine proteases kallikreins are associated with malignancy and may have potential diagnostic/prognostic applications in cancer. Kallikrein 6 (KLK6) is a member of the family of 15 highly conserved secreted trypsin- or chemotrypsin-like serine proteases. Overexpression of KLK6 has been observed in different pathophysiological states such as neurodegenerative diseases, inflammation and cancer. Activating K-RAS mutations are common in colon cancer progression and contribute to many cancerous phenotypes including increased proliferation, evasion of apoptosis, and increased migration and invasion. We have shown that acquisition of K-RAS mutations in colon cancer cells leads to overexpression of membrane protein caveolin 1 and results in increase of KLK6 expression and secretion. The aim of this study was to elucidate the role of KLK6 expression in metastatic CRC in relation to the status of K-RAS oncogene. Analysis of KLK6 expression in CRC patients by immunohistochemistry shows the statistically significant increase in the staining score for KLK6 protein in the microscopically normal tissue 10 cm away from tumors, which carry mutant K-RAS (G12A mutation) (p=0.05 for crypts staining and p=0.0445 for the surface area). These data suggest a specific role of KLK6 in the invasive phenotype associated with the presence of K-RAS mutation. To investigate the significance of KLK6 expression in metastatic CRC, we developed HCT116 human colon cancer isogenic cell lines (K-RAS G13D mutation) with the genetic knock down of KLK6 expression (shKLK6 clones). The shKLK6 clones had decreased expression and secretion of KLK6 protein with the minimal effect on the cell growth and viability in cell culture. SCID mice injected with shKLK6 cells exhibited a statistically significant increase in the survival rates (p=0.005), decrease in the incidence of distant metastases and the shift in the location of the metastatic foci closer to the cells injection site. Levels of KLK6 protein secreted into the bloodstream were significantly lower in animals injected with shKLK6 clone compared to HCT116 control clone (p
• Meuillet, E. J., Meuillet, E. J., Boros, L. G., Boros, L. G., Cantoria, M. J., Cantoria, M. J., Han, H., Han, H., Ignatenko, N. A., Ignatenko, N. A., Patel, H., & Patel, H. (2014). Abstract 1434: Metformin-induced metabolic changes are k-ras-dependent in animal models of pancreatic cancer. Cancer Research, 74(19_Supplement), 1434-1434. doi:10.1158/1538-7445.am2014-1434
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  Pancreatic ductal adenocarcinoma (PDA) is an aggressive malignancy, that is very difficult to treat. To date, systemic treatment for PDA, either a single agent or combinations of agents, has shown only modest benefits. Numerous epidemiological studies have reported that metformin (MET), a widely used anti-diabetic drug, provides protective benefits in reducing PDA risk among the diabetic population. Using a stable isotope glucose (GLUC) tracer for dynamic metabolic profiling (SiDMAP), we recently reported that high cholesterol (CHOL) alters the cellular metabolism of MiaPaCa2 (a PDA cell line harboring mutant K-Ras) cells by redirecting glucose-derived acetyl-CoA toward fatty acid (FA) synthesis. This response to MET is depended on the level of intracellular CHOL synthesis. We now performed a SiDMAP study in the LSL-K-RasG12D/+, LSL-Trp53R172H/+, Pdx-1-Cre (KPC) and the LSL-K-RasG12D/+, Pdx-1-Cre (KC) mouse models for PDA, and their wild-type littermates (C57Bl6.129). The mice were treated (or not) with MET (250 mg/kg, i.p. Q5D) and subjected to an Intra Peritoneal GLUC Tolerance Test (IPGTT) pre- and post-MET treatment. The KPC mice with the average tumor volume of 80.83+8.57 mm3 and the KC mice with PanIN lesions (aver. 9 mo old) were put on study. K-Ras mutation, with the presence of the tumor (KPC mice), induced an increase in plasma GLUC production via de novo synthesis by the liver using futile cycling of GLUC derived lactate and pyruvate. MET treatment decreased this flux in mutated (KC) and tumor-bearing animals (KPC), but increased this flux in liver of control mice. MET treatment increased the complete GLUC oxidation into 13CO2 in the pancreas of KC and KPC animals. This indicates that the pancreas in MET-treated animals use less GLUC for RNA, DNA and FA synthesis. For 13CO2 production, we found that tumor growth has to be established; the mutation is not enough to induce changes in this flux. As in plasma, tumor growth increases pancreas lactate production from GLUC. MET treatment dramatically decreased the Warburg effect in the pancreas of mutated (KC) and mutated/tumor-bearing (KPC) mice. Finally, MET decreased GLUC-derived acetyl-CoA delivery to FAS and palmitate in control, mutated and tumor-bearing mice. Immunohistochemical analysis of the tumors from the KPC mice revealed that treatment with MET decreased the staining of phospho-Ser79-ACC by ∼54%, total FAS staining (mainly in the tumor stroma) by ∼43%, and the growth-associated transcription factor HMGA2 by ∼36.7%. These decreases correlated with a decrease in Ki67 staining by almost 80%, indicative of an inhibitory effect of MET on PDA growth. These results suggest that mutant K-Ras is responsible for the metabolic adaptation in both the tumor- (KPC) and non-tumor-bearing animals (KC). Our findings provide a strong rationale for targeting the metabolic changes induced by activated K-RAS in PDA patients which harbor mutations in K-RAS gene in >95% of cases. Citation Format: Mary Jo Cantoria, Laszlo G. Boros, Hitendra Patel, Haiyong Han, Natalia Ignatenko, Emmanuelle J. Meuillet. Metformin-induced metabolic changes are k-ras-dependent in animal models of pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1434. doi:10.1158/1538-7445.AM2014-1434
• Banerjee, B., Mckeown, K. R., Skovan, B., Ogram, E., Ingram, P., Ignatenko, N. A., Paine-Murrieta, G., Witte, R., Matsunaga, T., Ignatenko, N. A., & Gerner, E. W. (2013). Ultrasound imaging of the mouse pancreatic duct using lipid microbubbles; Get the fat out!. Cancer prevention research (Philadelphia, Pa.), 832018; 6, 161-164.
• Gerner, E. W., & Ignatenko, N. A. (2013). Get the fat out!. Cancer prevention research (Philadelphia, Pa.), 6(3), 161-4. doi:10.1158/1940-6207.capr-13-0025
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  Obesity is associated with increased risk of a number of cancers in humans, but the mechanism(s) responsible for these associations have not been established. It is estimated that 68% of adults are overweight or obese and that obesity may be causative in 4% to 7% of cancers in the United States. Several hypotheses have been put forward to explain the association between obesity and cancer including adipose-directed signaling (e.g., mTOR, AMPK), production of factors (e.g., insulin growth factor 1, fibroblast growth factor 1, and/or chronic inflammation associated with obesity. Huffman and colleagues used surgical methods to determine if visceral fat was causally related to intestinal tumorigenesis in the Apc(1638/N+) mouse in a manner independent of confounding factors such as caloric restriction. They found that caloric restriction could extend survival in both male and female Apc(1638/N+) mice but found that surgical removal of visceral fat was only effective in reducing macroadenomas in females. The results of this study do not identify the specific mechanism of association between visceral fat and intestinal carcinogenesis in female mice but do support the rationale for future cancer prevention trials that evaluate pharmacologic and behavioral strategies to reduce abdominal obesity in humans. Cancer Prev Res; 6(3); 161-4. ©2013 AACR.
• Ignatenko, N., Basu Roy, U. K., Henkhaus, R. S., Loupakis, F., Cremolini, C., Gerner, E. W., & Ignatenko, N. -. (2013). Caveolin-1 is a novel regulator of K-RAS-dependent migration in colon carcinogenesis. International journal of cancer. Journal international du cancer, 133(1).
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  Caveolin-1 is an essential component of membrane caveolae. It is an important regulator of cellular processes such as signal transduction and endocytosis. We report here, for the first time, that caveolin-1 is a target of the K-RAS oncogene in colon carcinogenesis. Caveolin-1 is induced in colon cancer cells and in human colon tumor samples, in response to K-RAS activating mutations. An activated K-RAS oncogene transcriptionally induces caveolin-1 expression in human colon cancer cells and this effect is not restricted to the type of activating K-RAS mutation. Inhibition of the P-I3 Kinase-AKT pathway, but not the ERK MAPK pathway, both important K-RAS effectors, leads to a decrease in caveolin-1 expression indicating that the AKT pathway is involved in caveolin-1 expression in response to an activated K-RAS. Increased AKT signaling induces caveolin-1 expression by increasing the activity of the transcription factor, Sp1. Interestingly; caveolin-1 depletion alters K-RAS-dependent signaling by decreasing Grb2-SOS activity. Consistent with these finding, caveolin-1-depleted cells shows decreased migration in vitro. However, caveolin-1 overexpression by itself does not increase migration whereas an activated Src can increase migration in a caveolin-1-dependent manner. This increased migration is highly dependent on the RhoA GTPase, indicating that an activated K-RAS modulates migration in part via caveolin-1 induction, and increasing RhoA activity via phospho-caveolin-1. Our findings indicate that K-RAS regulates both caveolin-1 expression and other factors affecting caveolin-1 functions in colon cancer-derived cell migration.
• Matsunaga, T. O., Banerjee, B., Ignatenko, N. A., Ingram, P., Mckeown, K. R., Ogram, E., Paine-murrieta, G., Skovan, B. A., & Witte, R. S. (2012). Ultrasound imaging of the mouse pancreatic duct using lipid microbubbles. Proceedings of SPIE, 8320. doi:10.1117/12.902635
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  Research requiring the murine pancreatic duct to be imaged is often challenging due to the difficulty in selectively cannulating the pancreatic duct. We have successfully catheterized the pancreatic duct through the common bile duct in severe combined immune deficient (SCID) mice and imaged the pancreatic duct with gas filled lipid microbubbles that increase ultrasound imaging sensitivity due to exquisite scattering at the gas/liquid interface. A SCID mouse was euthanized by CO 2 , a midline abdominal incision made, the common bile duct cut at its midpoint, a 2 cm, 32 gauge tip catheter was inserted about 1 mm into the duct and tied with suture. The duodenum and pancreas were excised, removed in toto, embedded in agar and an infusion pump was used to instill normal saline or lipid-coated microbubbles (10 million / ml) into the duct. B-mode images before and after infusion of the duct with microbubbles imaged the entire pancreatic duct (~ 1 cm) with high contrast. The microbubbles were cavitated by high mechanical index (HMI) ultrasound for imaging to be repeated. Our technique of catheterization and using lipid microbubbles as a contrast agent may provide an effective, affordable technique of imaging the murine pancreatic duct; cavitation with HMI ultrasound would enable repeated imaging to be performed and clustering of targeted microbubbles to receptors on ductal cells would allow pathology to be localized accurately. This research was supported by the Experimental Mouse Shared Service of the AZ Cancer Center (Grant Number P30CA023074, NIH/NCI and the GI SPORE (NIH/NCI P50 CA95060).
• Ignatenko, N. A., Gerner, E. W., & Besselsen, D. G. (2011). Defining the role of polyamines in colon carcinogenesis using mouse models. Journal of carcinogenesis, 10, 10-3163.79673. Epub 2011 Apr 16.
• Paz, E. A., Garcia-Huidobro, J., & Ignatenko, N. A. (2011). Polyamines in cancer. Advances in Clinical Chemistry, 54, 45-70.
• Ignatenko, N. A., Yerushalmi, H. F., Pandey, R., Kachel, K. L., Stringer, D. E., Marton, L. J., & Gerner, E. W. (2009). Gene expression analysis of HCT116 colon tumor-derived cells treated with the polyamine analog PG-11047. Cancer genomics & proteomics, 6, 161-175.
• Zell, J. A., Ziogas, A., Ignatenko, N., Honda, J., Qu, N., Bobbs, A. S., Neuhausen, S. L., Gerner, E. W., & Anton-Culver, H. (2009). Associations of a polymorphism in the ornithine decarboxylase gene with colorectal cancer survival. Clinical cancer research : an official journal of the American Association for Cancer Research, 15, 6208-6216.
• Henkhaus, R. S., Gerner, E. W., & Ignatenko, N. A. (2008). Kallikrein 6 is a mediator of K-RAS-dependent migration of colon carcinoma cells. Biological chemistry, 389, 757-764.
• Henkhaus, R. S., Roy, U. K., Cavallo-Medved, D., Sloane, B. F., Gerner, E. W., & Ignatenko, N. A. (2008). Caveolin-1-mediated expression and secretion of kallikrein 6 in colon cancer cells. Neoplasia (New York, N.Y.), 10, 140-148.
• Ignatenko, N. A., & Gerner, E. W. (2008). HuRrying colon cancer progression. Cancer biology & therapy, 7, 428-429.
• Ignatenko, N. A., Besselsen, D. G., Stringer, D. E., Blohm-Mangone, K. A., Cui, H., & Gerner, E. W. (2008). Combination chemoprevention of intestinal carcinogenesis in a murine model of familial adenomatous polyposis. Nutrition and cancer, 60 Suppl 1, 30-35.
• Ignatenko, N., Henkhaus, R. S., Gerner, E. W., & Ignatenko, N. -. (2008). Kallikrein 6 is a mediator of K-RAS-dependent migration of colon carcinoma cells. Biological chemistry, 389(6).
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  Kallikrein 6 (KLK6) is a trypsin-like serine peptidase whose relevance in various types of cancers is currently being explored. Previous studies have shown that KLK6 mRNA is upregulated in colon and gastric cancers; however, the regulatory mechanisms and phenotypic consequences of this upregulation are largely unknown. Activating K-RAS mutations are common in colon cancer, occurring in approximately 50% of cases. We have recently reported the upregulation of KLK6 mRNA in Caco2 human colon cancer cells stably transfected with a mutant K-RAS allele (K-RAS(G12V)). In this study we examined the pattern of K-RAS-dependent KLK6 expression and secretion in colon cancer cells. Using pharmacological inhibitors of pathways downstream of K-RAS, we could show that the PI3K and p42/44 MAPK pathways play an important role in the induction of KLK6 in mutant K-RAS-expressing colon cancer cells. Increased KLK6 expression enhanced colon cancer cell migration through laminin and Matrigel. Inhibition of KLK6 using small interference RNA treatment or a specific KLK6 antibody in Caco2 cells stably expressing the mutant K-RAS and in SW480 cells carrying a mutation in the K-RAS oncogene resulted in a reduction in invasiveness through cell culture inserts. These data support the oncogenic role of KLK6 in colorectal cancer.
• Ignatenko, N., Henkhaus, R. S., Roy, U. K., Cavallo-Medved, D., Sloane, B. F., Gerner, E. W., & Ignatenko, N. -. (2008). Caveolin-1-mediated expression and secretion of kallikrein 6 in colon cancer cells. Neoplasia (New York, N.Y.), 10(2).
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  Kallikreins are secreted proteases that may play a functional role and/or serve as a serum biomarker for the presence or progression of certain types of cancers. Kallikrein 6 (KLK6) has been shown to be upregulated in several types of cancers, including colon. The aims of this study were to elucidate pathways that influence KLK6 gene expression and KLK6 protein secretion in the HCT116 human colon cancer cells. Our data indicate a central role for caveolin-1 (CAV-1), the main structural protein of caveolae, in both KLK6 gene expression and protein secretion. Sucrose gradient subcellular fractionation reveals that CAV-1 and KLK6 colocalize to lipid raft domains in the plasma membrane of HCT116 cells. Furthermore, we show that CAV-1, although it does not directly interact with the KLK6 molecule, enhances KLK6 secretion from the cells. Deactivation of CAV-1, through SRC-mediated phosphorylation, decreased KLK6 secretion. We also demonstrate that, in colon cancer cells, CAV-1 increased the amount of phosphorylated AKT in cells by inhibiting the activity of the AKT-negative regulators PP1 and PP2A. This study demonstrates that proteins such as CAV-1 and AKT, which are known to be altered in colon cancer, affect KLK6 expression and KLK6 secretion.
• Roy, U. K., Henkhaus, R. S., Ignatenko, N. A., Mora, J., Fultz, K. E., & Gerner, E. W. (2008). Wild-type APC regulates caveolin-1 expression in human colon adenocarcinoma cell lines via FOXO1a and C-myc. Molecular carcinogenesis, 47, 947-955.
• Bernstein, H., Holubec, H., Bernstein, C., Ignatenko, N. A., Gerner, E., Dvorak, K., Besselsen, D., Blohm-Mangone, K. A., Padilla-Torres, J., Dvorakova, B., Garewal, H., & Payne, C. M. (2007). Deoxycholate-induced colitis is markedly attenuated in Nos2 knockout mice in association with modulation of gene expression profiles. Digestive diseases and sciences, 52, 628-642.
• Hariri, L. P., Qiu, Z., Tumlinson, A. R., Besselsen, D. G., Gerner, E. W., Ignatenko, N. A., Povazay, B., Hermann, B., Sattmann, H., McNally, J., Unterhuber, A., Drexler, W., & Barton, J. K. (2007). Serial endoscopy in azoxymethane treated mice using ultra-high resolution optical coherence tomography. Cancer biology & therapy, 6, 1753-1762.
• Zell, J. A., Ignatenko, N. A., Yerushalmi, H. F., Ziogas, A., Besselsen, D. G., Gerner, E. W., & Anton-Culver, H. (2007). Risk and risk reduction involving arginine intake and meat consumption in colorectal tumorigenesis and survival. International journal of cancer.Journal international du cancer, 120, 459-468.
• Bernstein, H., Holubec, H., Bernstein, C., Ignatenko, N., Gerner, E., Dvorak, K., Besselsen, D., Ramsey, L., Dall'Agnol, M., Blohm-Mangone, K. A., Padilla-Torres, J., Cui, H., Garewal, H., & Payne, C. M. (2006). Unique dietary-related mouse model of colitis. Inflammatory bowel diseases, 12, 278-293.
• Ignatenko, N. A., Besselsen, D. G., Roy, U. K., Stringer, D. E., Blohm-Mangone, K. A., Padilla-Torres, J. L., Guillen-R, J. M., & Gerner, E. W. (2006). Dietary putrescine reduces the intestinal anticarcinogenic activity of sulindac in a murine model of familial adenomatous polyposis. Nutrition and cancer, 56, 172-181.
• Ignatenko, N. A., Holubec, H., Besselsen, D. G., Blohm-Mangone, K. A., Padilla-Torres, J. L., Nagle, R. B., Alboranc, I. M., Guillen-R, J. M., & Gerner, E. W. (2006). Role of c-Myc in intestinal tumorigenesis of the ApcMin/+ mouse. Cancer biology & therapy, 5, 1658-1664.
• Ignatenko, N. A., Yerushalmi, H. F., Watts, G. S., Futscher, B. W., Stringer, D. E., Marton, L. J., & Gerner, E. W. (2006). Pharmacogenomics of the polyamine analog 3,8,13,18-tetraaza-10,11-[(E)-1,2-cyclopropyl]eicosane tetrahydrochloride, CGC-11093, in the colon adenocarcinoma cell line HCT1161. Technology in cancer research & treatment, 5, 553-564.
• Yerushalmi, H. F., Besselsen, D. G., Ignatenko, N. A., Blohm-Mangone, K. A., Padilla-Torres, J. L., Stringer, D. E., Cui, H., Holubec, H., Payne, C. M., & Gerner, E. W. (2006). The role of NO synthases in arginine-dependent small intestinal and colonic carcinogenesis. Molecular carcinogenesis, 45, 93-105.
• Yerushalmi, H. F., Besselsen, D. G., Ignatenko, N. A., Blohm-Mangone, K. A., Padilla-Torres, J. L., Stringer, D. E., Guillen, J. M., Holubec, H., Payne, C. M., & Gerner, E. W. (2006). Role of polyamines in arginine-dependent colon carcinogenesis in Apc(Min) (/+) mice. Molecular carcinogenesis, 45, 764-773.
• Gerner, E. W., Besselsen, D. G., Blohm-mangone, K. A., Dexter, E. I., Holubec, H., Ignatenko, N. A., Nagle, R. B., & Padilla-tores, J. L. (2005). Role of c-MYC in normal intestinal development and tumorigenesis in the ApcMin/+ mouse. Cancer Research, 65, 464-464.
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  Proc Amer Assoc Cancer Res, Volume 46, 2005 1982 The c-Myc oncogene plays an important role in tumorigenesis and is commonly activated in gastrointestinal cancers. In human colon tumor cells, c-Myc is regulated by the adenomatous polyposis coli (APC) tumor suppressor gene, which is mutated or deleted in humans with familial adenomatous polyposis (FAP). The ApcMin/+ (Min) mice are a model of FAP and are heterozygous for a truncating Apc mutation. We have developed a mouse model for studying the role of c-myc in the mutant APC-mediated colon tumorigenesis. Mice harboring a transgene containing the Cre recombinase under the control of transcriptional regulatory elements from a fatty acid-binding protein gene ( Fabp 4×at132) were crossed with mice homozygous for the c-mycflox allele. Progeny were then interbred to initiate the deletion of loxP flanked c-myc sequences in rapidly self-renewing mouse small intestinal and colonic epithelium. Cre expression and recombination in the transgenic c-myc null mouse ( Fabp 4×at 132 Cre transgenic mice x c-myc null) resulted in mosaic loss of the expression of the c-myc protein in ileum, cecum and colon tissues, in which the Fabp4×at 132 promoter is active. Conditional c-myc null mice exhibited underdeveloped intestinal crypts and villous atrophy. The results of recombination were evaluated microscopically using methylene-blue stained whole-mount preparations and were confirmed by morphology in H&E stained sections and by immunohistochemical analysis using c-MYC antibody. We crossed the Fabp4×at 132Cre c-myc null mouse to ApcMin/+ and then to their progeny - to create the ApcMin/+ mouse with intestinal specific knockout of the c-myc oncogene ( ApcMin/+ c-myc null). The compound ApcMin/+ c-myc null mouse was used to evaluate the effect of colon-specific c-myc knockout on the ApcMin/+ phenotype. We observed a relative reduction in the tumor number in the small intestine in ApcMin/+ c-myc conditional knockout mice compared to control ApcMin/+ mice. These data demonstrate that c-Myc is essential for normal development of intestinal epithelia and impacts either directly or indirectly Apc-dependent intestinal tumorigenesis in mice.
• Gerner, E. W., Ignatenko, N. A., Lance, P., & Hurley, L. H. (2005). A comprehensive strategy to combat colon cancer targeting the adenomatous polyposis coli tumor suppressor gene. Annals of the New York Academy of Sciences, 1059, 97-105.
• Gerner, E. W., Besselsen, D. G., Blohm-mangone, K. A., Holubec, H., Ignatenko, N. A., Padilla-torres, J. L., Payne, C. M., Yamauchi, P., Yerushalmi, H. F., & Zhang, H. (2004). Gene-diet interactions in arginine-dependent intestinal carcinogenesis. Cancer Research, 64, 900-900.
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  3904 L-arginine, an essential amino acid, is converted by arginase to ornithine, the substrate for polyamine synthesis. Polyamines (putrescine, spermidine and spermine) are small ubiquitous molecules involved in various processes and required for cell proliferation. L-arginine is also metabolized by nitric oxide synthases (NOSs) to form nitric oxide (NO) that can influence signal transduction and DNA damage. NOSs are found as constitutively expressed isoforms (NOS1/NOS3) or the inducible isoform (NOS2). In this study, we evaluated NOS2 and dietary L-arginine effects on intestinal tumorgenesis in Min (multiple intestinal neoplasia) mice. Min mice were crossed with NOS2 knockout mice or wild-type NOS2 mice. NOS2 protein was expressed in small intestine, but not the colon, of Min X NOS2 (+/+) mice. The protein was not detected in either intestinal tissue of Min X NOS2 (−/−) mice. When animals were fed an arginine-restricted diet, loss of NOS2 alleles was associated with a small (50% or less) increase in small intestinal tumor number and colonic tumor incidence, compared to Min X NOS2 (+/+) mice. Supplemental arginine (up to 2% in the drinking water) slightly decreased small intestinal tumor number, in a concentration-dependent manner, in Min X NOS2 (−/−) mice. In mice expressing NOS2, intestinal tumor number was minimally affected by dietary arginine. In these mice, dietary arginine increased small intestinal arginine and ornithine contents, whereas in Min X NOS2 (−/−) mice no changes were observed. Colon tumor incidence showed an increase with dietary arginine, independent of NOS2 expression. This increase can result from dietary-arginine dependent increase in ornithine and putrescine contents. These data suggest that NOS2 expression and dietary arginine have only minimal effects on small intestinal carcinogenesis in the Min mouse. However, dietary arginine increases the incidence of colonic tumors, resulting from lack of expression of the adenomatous polyposis coli (APC) gene, by a NOS2-independent mechanism that may involve polyamines.
• Ignatenko, N. A., Babbar, N., Mehta, D., Jr, R. A., & Gerner, E. W. (2004). Suppression of polyamine catabolism by activated Ki-ras in human colon cancer cells. Molecular carcinogenesis, 39, 91-102.
• Ignatenko, N. A., Zhang, H., Watts, G. S., Skovan, B. A., Stringer, D. E., & Gerner, E. W. (2004). The chemopreventive agent alpha-difluoromethylornithine blocks Ki-ras-dependent tumor formation and specific gene expression in Caco-2 cells. Molecular carcinogenesis, 39, 221-233.
• Zaletok, S., Alexandrova, N., Berdynskykh, N., Ignatenko, N., Gogol, S., Orlovsky, O., Tregubova, N., Gerner, E., & Chekhun, V. (2004). Role of polyamines in the function of nuclear transcription factor NF-kappaB in breast cancer cells. Experimental oncology, 26, 221-225.
• Babbar, N., Ignatenko, N. A., Jr, R. A., & Gerner, E. W. (2003). Cyclooxygenase-independent induction of apoptosis by sulindac sulfone is mediated by polyamines in colon cancer. The Journal of biological chemistry, 278, 47762-47775.
• Gerner, E. W., Besselsen, D. G., & Ignatenko, N. A. (2003). Preclinical models for chemoprevention of colon cancer.. Recent results in cancer research. Fortschritte der Krebsforschung. Progres dans les recherches sur le cancer, 163, 58-71; discussion 264-6. doi:10.1007/978-3-642-55647-0_6
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  Colon cancer is the second leading cause of cancer incidence and death in the USA in 2002. Specific genetic defects have been identified which cause hereditary colon cancers in humans. In addition, a number of intestinal luminal risk factors for colon cancer have been described. This information has been exploited to develop experimental cell and rodent models which recapitulate features of human colon cancer. In this chapter, we will discuss the strengths and limitations of these models to further our understanding of basic mechanisms of colon carcinogenesis and to develop strategies for colon cancer chemoprevention.
• Gerner, E. W., Broome-Powell, M., Erdman, S. H., Ignatenko, N. A., Besselsen, D. G., Gerner, E. W., Ignatenko, N. A., Fultz, K. E., Gerner, E. W., Ignatenko, N. A., & Besselsen, D. G. (2003). COST 917: Biogenically Active Amines in Food; Pharmaceutical Perspectives of Nucleic Acid-Based Therapeutics; Preclinical models for chemoprevention of colon cancer. Recent results in cancer research.Fortschritte der Krebsforschung.Progres dans les recherches sur le cancer, 163, 58-71; discussion 264-6.
• Gerner, E. W., Fultz, K. E., Ignatenko, N. A., Martinez, J. D., & Parker, M. T. (2003). Molecular Biology of Cancer. Burger's Medicinal Chemistry and Drug Discovery, 1-50. doi:10.1002/0471266949.bmc074
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  Over the past 50 years, our understanding of cancer has grown dramatically. Conceptual models that explain the tumor development process have been formulated. The biological characteristics that account for the behavior of tumor cells in experimental systems and in vivo and the genetic alterations that account for these changes have been catalogued in detail. Additionally, new preventative strategies and therapeutic agents using this wealth of information are now being formulated. In this chapter, we present a brief overview of our current understanding of cancer as a disease as well as give specific examples of some of the approaches being adopted to combat it. Keywords: tumorigenesis; cancer; apoptosis; angiogenesis; oncogene; tumor suppressor gene; gene therapy; cell cycle
• Martinez, J. D., Parker, M. T., Fultz, K. E., Ignatenko, N. A., Gerner, E. W., Ignatenko, N. A., & Gerner, E. W. (2003). Burger's Medicinal Chemistry and Drug Discovery; Regulation of the HIV1 long terminal repeat by mutant heat shock factor. Experimental cell research, 288, 1156; 1-8.
• Qu, N., Ignatenko, N. A., Yamauchi, P., Stringer, D. E., Levenson, C., Shannon, P., Perrin, S., & Gerner, E. W. (2003). Inhibition of human ornithine decarboxylase activity by enantiomers of difluoromethylornithine. The Biochemical journal, 375, 465-470.
• Gerner, E. W., Besselsen, D. G., & Ignatenko, N. A. (2002). Session 4 S9. Genetically altered rodents and human tumor cells as preclinical models for prevention of intestinal cancers. European Journal of Cancer, 38, S12. doi:10.1016/s0959-8049(02)80617-7
• Lawson, K. R., Ignatenko, N. A., Piazza, G. A., Cui, H., & Gerner, E. W. (2000). Influence of K-ras activation on the survival responses of Caco-2 cells to the chemopreventive agents sulindac and difluoromethylornithine. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology, 9, 1155-1162.
• Taylor, M. T., Lawson, K. R., Ignatenko, N. A., Marek, S. E., Stringer, D. E., Skovan, B. A., & Gerner, E. W. (2000). Sulindac sulfone inhibits K-ras-dependent cyclooxygenase-2 expression in human colon cancer cells. Cancer research, 60, 6607-6610.
• Erdman, S. H., Ignatenko, N. A., Powell, M. B., Blohm-Mangone, K. A., Holubec, H., Guillen-Rodriguez, J. M., & Gerner, E. W. (1999). APC-dependent changes in expression of genes influencing polyamine metabolism, and consequences for gastrointestinal carcinogenesis, in the Min mouse. Carcinogenesis, 20, 1709-1713.
• Gerner, E. W., & Ignatenko, N. A. (1996). Growth arrest- and polyamine-dependent expression of spermidine/spermine N1-acetyltransferase in human tumor cells.. Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research, 7(4), 481-6.
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  Polyamines are essential for optimal cell growth. The regulation of polyamine biosynthetic, but not catabolic, enzymes has been studied in detail. Because intracellular polyamine contents depend on both synthesis and catabolism, we studied the regulation of spermidine/spermine N1-acetyltransferase (N1SSAT), the first enzyme in polyamine catabolism. Steady-state RNA levels of N1SSAT increased 3-5 fold as human colon tumor-derived HCT116 cells traversed the log phase and entered the plateau phase. Depletion of cellular polyamines, using alpha-difluoromethylornithine, caused a decrease in the steady-state levels of both the 1.3-kb N1SSAT transcript and its 3.5-kb precursor, without affecting the stability of either RNA. N1SSAT enzyme activity was low in cells with normal polyamine contents but could be induced by heat shock. The level of induction of N1SSAT enzyme activity by heat shock on different days of growth correlated with N1SSAT RNA levels prior to heat shock and occurred without changes in levels of message after heat shock. Although non-heat-shocked cells containing normal polyamine contents expressed N1SSAT RNA but not enzyme activity, exogenous spermidine restored both RNA levels and enzyme activity in polyamine-depleted cells. This result suggests that the expression of N1SSAT enzyme activity, but not RNA, requires a change in the intracellular compartmentalization of spermidine. These data demonstrate that N1SSAT is regulated at both the transcriptional and posttranscriptional levels by conditions that arrest growth in HCT116 cells, and that both of these mechanisms are affected by endogenous polyamine contents.
• Ignatenko, N. A., Fish, J. L., Shassetz, L. R., Woolridge, D. P., & Gerner, E. W. (1996). Expression of the human spermidine/spermine N1-acetyltransferase in spermidine acetylation-deficient Escherichia coli. The Biochemical journal, 319 ( Pt 2), 435-440.
• Ignatenko, N., & Gerner, E. (1996). Growth arrest- and polyamine-dependent expression of spermidine/spermine N1-acetyltransferase in human tumor cells. Cell Growth and Differentiation, 7(4).
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  Polyamines are essential for optimal cell growth. The regulation of polyamine biosynthetic, but not catabolic, enzymes has been studied in detail. Because intracellular polyamine contents depend on both synthesis and catabolism, we studied the regulation of spermidine/spermine N1- acetyltransferase (N1SSAT), the first enzyme in polyamine catabolism. Steady-state RNA levels of N1SSAT increased 3-5 fold as human colon tumor- derived HCT116 cells traversed the log phase and entered the plateau phase. Depletion of cellular polyamines, using α-difluoromethylornithine, caused a decrease in the steady-state levels of both the 1.3-kb N1SSAT transcript and its 3.5-kb precursor, without affecting the stability of either RNA. N1SSAT enzyme activity was low in cells with normal polyamine contents but could be induced by heat shock. The level of induction of N1SSAT enzyme activity by heat shock on different days of growth correlated with N1SSAT RNA levels prior to heat shock and occurred without changes in levels of message after heat shock. Although non-heat-shocked cells containing normal polyamine contents expressed N1SSAT RNA but not enzyme activity, exogenous spermidine restored both RNA levels and enzyme activity in polyamine-depleted cells. This result suggests that the expression of N1SSAT enzyme activity, but not RNA, requires a change in the intracellular compartmentalization of spermidine. These data demonstrate that N1SSAT is regulated at both the transcriptional and posttranscriptional levels by conditions that arrest growth in HCT116 cells, and that both of these mechanisms are affected by endogenous polyamine contents.
• Zaletok, S. P., Lyalyushko, N. M., Berdinskikh, N. K., Ignatenko, N. A., Lidak, M. Y., Baumanis, E. A., Birska, I. A., Zaletok, S. P., Berdinskikh, N. K., Ignatenko, N. A., Baumanis, E. A., Lidak, M. Y., Lyalyushko, N. M., Ignatenko, N. A., & Gerner, E. W. (1996). The influence of polyhexamethyleneguanidine on polyamine methabolism in mongrel rats and on the growth and metastatic spreading of transplantable Lewis lung carcinoma in C57B1 mice; The effect of polyhexamethyleneguanidine on some indices of polyamine metabolism in rats with transplantable hepatoma H-27; Growth arrest- and polyamine-dependent expression of spermidine/spermine N1-acetyltransferase in human tumor cells. Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research, 15; 16; 7, 36; 136; 481-41; 139; 486.
• Zaletok, S., Berdinskikh, N., Ignatenko, N., Baumanis, E., Lidak Yu., M., & Lyalyushko, N. (1994). The effect of polyhexamethyleneguanidine on some indices of polyamine metabolism in rats with transplantable hepatoma H-27. Eksperimentalnaya Onkologiya, 16(2-3).
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  The influence of polyhexamethyleneguanidine (PMG) on some indices of polyamine metabolism in liver and tumor tissue of rats with transplantable hepatoma H-27 as well as the plasma membrane fraction of these cells was studied. It has been established that ornithindecarboxylase (ODC) activity and polyamine content in hepatoma tissue considerably exceeded those one in the liver of tumorbearing rats and in the liver of intact animals. The ODC activity and polyamine level in liver and tumor tissue decreased after PMG administration. Under the chosen regime of PMG administration 2-fold decrease of ODC activity and the decrease of polyamine level along with 3-fold increase in γ-glutamyltranspeptidase activity were observed. Possible mechanisms of PMG antitumor effect are discussed.
• Zaletok, S., Lyalyushko, N., Berdinskikh, N., Ignatenko, N., Lidak Yu., M., Baumanis, E., & Birska, I. (1993). The influence of polyhexamethyleneguanidine on polyamine metabolism in mongrel rats and on the growth and metastatic spreading of transplantable Lewis lung carcinoma in C57Bl mice. Eksperimentalnaya Onkologiya, 15(6).
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  Polyhexamethyleneguanidine (PMG) has been studied for its effect on some indices of polyamine metabolism in mongrel rats after preliminary induction of ornithine decarboxylase (ODC) activity in kidneys by means of testosterone. It has been found that PMG influences the synthesis of polyamines and inhibits activity of ODC, a key enzyme of their synthesis, which causes a decrease of the polyamines content in the target organ and in biological fluids of the organism (blood and urine). The effect of PMG on the growth and metastatic spreading of the Lewis lung carcinoma was studied in C57Bl mice. It has been shown that antitumour and antimetastatic effect of PMG depends on the dose, the number of injections and the terms of the drug administration. Combined administration of ceruloplasmin and PMG reduces the toxic effect of the latter and intensifies the antitumour and antimetastatic effects, which evidences for the expediency of combined application of PMG and ceruloplasmin.
• Berdinskikh, N. K., Ignatenko, N. A., Zaletok, S. P., Ganina, K. P., & Chorniy, V. A. (1991). Ornithine decarboxylase activity and polyamine content in adenocarcinomas of human stomach and large intestine. International journal of cancer.Journal international du cancer, 47, 496-498.
• Berdinskikh, N. K., Ignatenko, N. A., & Zaletok, S. P. (1990). Ornithine decarboxylase activity and polyamine contents in 1,2-dimethylhydrazine-induced carcinogenesis of the intestines in rats. Eksperimental'naia onkologiia, 12, 34-36.
• Nk, B., Ignatenko, N. A., & Sp, Z. (1990). Aktivnost' ornitindekarboksilazy i soderzhanie poliaminov pri indutsirovannom 1,2-dimetilgidrazinom kantserogeneze kishechnika u krys.. Eksperimentalnaa onkologia, 10, 39-41.
• Berdinskikh, N. K., Zaletok, S. P., Bobro, L. I., & Ignatenko, N. A. (1988). Ornithine decarboxylase activity and polyamine content in human adenocarcinomas of the stomach and large intestine. Eksperimental'naia onkologiia, 10, 39-41.
• Berdinskikh, N. K., Zaletok, S. P., Ignatenko, N. A., & Draga, N. V. (1988). Increase in ornithine decarboxylase activity and polyamine levels of the rat liver during an early period of hepatocarcinogenesis. Ukrainskii biokhimicheskii zhurnal (1978), 60, 94-97.
• Nk, B., Sp, Z., Bobro, L. I., & Ignatenko, N. A. (1988). Aktivnost' ornitindekarboksilazy i soderzhanie poliaminov v adenokartsinomakh zheludka i tolstogo kishechnika cheloveka.. Eksperimental"naia onkologiia.