Proceedings of the 2022 National Toxicology Program Satellite Symposium.

The 2022 annual National Toxicology Program Satellite Symposium, entitled "Pathology Potpourri," was held in Austin, Texas at the Society of Toxicologic Pathology's 40th annual meeting during a half-day session on Sunday, June 19. The goal of this symposium was to present and discuss challenging diagnostic pathology and/or nomenclature issues. This article presents summaries of the speakers' talks along with select images that were used by the audience for voting and discussion. Various lesions and topics covered during the symposium included induced and spontaneous neoplastic and nonneoplastic lesions in the mouse lung, spontaneous lesions in the reproductive tract of a female cynomolgus macaque, induced vascular lesions in a mouse asthma model and interesting case studies in a rhesus macaque, dog and genetically engineered mouse model.

Gastric Neuroendocrine Tumors With Parietal Cell Atrophy in a Long-term Carcinogenicity Study in Rats.

Malignant neuroendocrine tumors were diagnosed in the stomach of two out of sixty female Sprague-Dawley rats treated for 89 weeks with a high dose of a novel, small molecule, cannabinoid-1 antagonist. The tumors were associated with parietal cell atrophy accompanied by foveolar hyperplasia of the glandular stomach mucosa. Parietal cell atrophy/foveolar hyperplasia was considered test article related at the high dose, given the higher incidence and severity relative to untreated controls, although the precise mechanism of the parietal cell atrophy was undetermined. Spontaneous gastric neuroendocrine tumors are very rare in rats, and the current cases were considered secondary to parietal cell atrophy causing reduced gastric acid secretion and subsequent overstimulation of gastrin release through a feedback loop.

Proceedings of the 2021 National Toxicology Program Satellite Symposium.

The 2021 annual National Toxicology Program (NTP) Satellite Symposium, entitled "Pathology Potpourri," was the 20th anniversary of the symposia and held virtually on June 25th, in advance of the Society of Toxicologic Pathology's 40th annual meeting. The goal of this symposium was to present and discuss challenging diagnostic pathology and/or nomenclature issues. This article presents summaries of the speakers' talks along with select images that were presented to the audience for voting and discussion. Various lesions and topics covered during the symposium included differentiation of canine oligodendroglioma, astrocytoma, and undefined glioma with presentation of the National Cancer Institute's updated diagnostic terminology for canine glioma; differentiation of polycystic kidney, dilated tubules and cystic tubules with a discussion of human polycystic kidney disease; a review of various rodent nervous system background lesions in control animals from NTP studies with a focus on incidence rates and potential rat strain differences; vehicle/excipient-related renal lesions in cynomolgus monkeys with a discussion on the various cyclodextrins and their bioavailability, toxicity, and tumorigenicity; examples of rodent endometrial tumors including intestinal differentiation in an endometrial adenocarcinoma that has not previously been reported in rats; a review of various rodent adrenal cortex lesions including those that represented diagnostic challenges with multiple processes such as vacuolation, degeneration, necrosis, hyperplasia, and hypertrophy; and finally, a discussion of diagnostic criteria for uterine adenomyosis, atypical hyperplasia, and adenocarcinoma in the rat.

Cooperation Between Distinct Cancer Driver Genes Underlies Intertumor Heterogeneity in Hepatocellular Carcinoma.

BACKGROUND AND AIMS: The pattern of genetic alterations in cancer driver genes in patients with hepatocellular carcinoma (HCC) is highly diverse, which partially explains the low efficacy of available therapies. In spite of this, the existing mouse models only recapitulate a small portion of HCC inter-tumor heterogeneity, limiting the understanding of the disease and the nomination of personalized therapies. Here, we aimed at establishing a novel collection of HCC mouse models that captured human HCC diversity. METHODS: By performing hydrodynamic tail-vein injections, we tested the impact of altering a well-established HCC oncogene (either MYC or ß-catenin) in combination with an additional alteration in one of eleven other genes frequently mutated in HCC. Of the 23 unique pairs of genetic alterations that we interrogated, 9 were able to induce HCC. The established HCC mouse models were characterized at histopathological, immune, and transcriptomic level to identify the unique features of each model. Murine HCC cell lines were generated from each tumor model, characterized transcriptionally, and used to identify specific therapies that were validated in vivo. RESULTS: Cooperation between pairs of driver genes produced HCCs with diverse histopathology, immune microenvironments, transcriptomes, and drug responses. Interestingly, MYC expression levels strongly influenced ß-catenin activity, indicating that inter-tumor heterogeneity emerges not only from specific combinations of genetic alterations but also from the acquisition of expression-dependent phenotypes. CONCLUSIONS: This novel collection of murine HCC models and corresponding cell lines establishes the role of driver genes in diverse contexts and enables mechanistic and translational studies.

Reactive oxygen species-mediated synergistic and preferential induction of cell death and reduction of clonogenic resistance in breast cancer cells by combined cisplatin and FK228.

Safe and effective combination chemotherapy regimens against breast cancer are lacking. We used our cellular system, consisting of the non-cancerous human breast epithelial MCF10A cell line and its derived tumorigenic, oncogenic H-Ras-expressing, MCF10A-Ras cell line, to investigate the effectiveness of a combination chemotherapy regimen in treating breast cancer cells using two FDA-approved agents, cisplatin and FK228. Cisplatin and FK228 significantly, synergistically, and preferentially induced death and reduced drug resistance of MCF10A-Ras versus MCF10A cells. The ERK-Nox-ROS pathway played a major role in both synergistic cell death induction and GSH-level reduction, which contributed to the synergistic suppression of drug resistance in cells. Enhancement of the Ras-ERK-Nox pathway by combined cisplatin and FK228 significantly increased ROS levels, leading to induction of death, reduction of drug resistance, and induction of DNA damage and oxidation in cancerous MCF10A-Ras cells. Furthermore, synergistic induction of cell death and reduction of drug resistance by combined cisplatin and FK228 in breast cells is independent of their estrogen receptor status. Our study suggests that combined cisplatin and FK228 should be considered in clinical trials as a new regimen for therapeutic control of breast cancers.

SMALL INTESTINAL ADENOCARCINOMA WITH CARCINOMATOSIS IN A SWIFT FOX (VULPES VELOX).

A 7-yr-old, intact, female swift fox (Vulpes velox) presented to the Veterinary Health Center at Kansas State University with a history of chronic weight loss, lethargy, inappetence, and myiasis. On physical examination, a firm mass was palpated in the mid- to cranial abdomen. The fox was euthanatized as a result of the grave prognosis. Gross necropsy and histologic findings included a small intestinal adenocarcinoma with diffuse transperitoneal spread throughout the abdominal cavity (carcinomatosis). To the authors' knowledge, this is the first report of intestinal adenocarcinoma with carcinomatosis in a swift fox.

CUTANEOUS EPITHELIOTROPIC T-CELL LYMPHOMA WITH METASTASES IN A VIRGINIA OPOSSUM (DIDELPHIS VIRGINIANA).

A 2-yr-old, captive, intact female Virginia opossum ( Didelphis virginiana ) with a 7-mo history of ulcerative dermatitis and weight loss was euthanatized for progressive worsening of clinical signs. Initially the opossum was treated with several courses of antibiotics, both topically and systemically; systemic nonsteroidal anti-inflammatory medication; and, later, systemic glucocorticoids, with no improvement in clinical signs. Histopathologic samples of skin lesions taken 3 mo into the course of disease revealed no evidence of neoplasia; however, cytologic samples of a skin lesion taken 5 mo into the course of disease revealed mature lymphocytes, and were suggestive of cutaneous lymphoma. Postmortem histopathology revealed neoplastic cells consistent with lymphoma; these were found in the haired skin of the forearm, axilla, hind limb, face, and lateral body wall, as well as the liver, kidney, axillary lymph node, heart, and spleen. Multifocal neutrophilic and eosinophilic ulcerative and necrotizing dermatitis and folliculitis of the haired skin were also present. To the authors' knowledge, this is the first documented case of cutaneous lymphoma in a Virginia opossum and the first documented case with visceral metastases in a marsupial.

Dipyridamole intervention of breast cell carcinogenesis.

Dietary prevention is a cost-efficient strategy to reduce the risk of human cancers. More than 85% breast cancers are sporadic and attributable to long-term exposure to environmental carcinogens through a multistep and multiyear disease process. We used our chronically induced cellular carcinogenesis model as a target to search for preventive agents capable of blocking breast cell carcinogenesis. Dipyridamole (DPM), at a non-cytotoxic physiologically achievable dose of 10 nmol/L, effectively blocked breast cell carcinogenesis induced by cumulative exposures to three unrelated carcinogens 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), benzo[a]pyrene (B[a]P), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). The ability of DPM to block H-Ras upregulation, thus blocking ERK pathway activation, reactive oxygen species (ROS) elevation, and DNA damage in each exposure, may account for its mechanisms in intervention of carcinogenesis induced by cumulative exposures to PhIP. Likewise, DPM's ability to block ROS elevation and DNA damage may account for its mechanisms in intervention of carcinogenesis chronically induced by NNK and B[a]P, as well. DPM is approved by the Food and Drug Administration to control platelet aggregation and vasoconstriction in patients. Our study revealed, for the first time, the novel ability of DPM to block breast cell carcinogenesis induced by three unrelated carcinogens. DPM should be seriously considered as a chemopreventive agent in development of strategies for reducing the risk of sporadic breast cancer associated with long-term exposure to environmental carcinogens.

Induction of human breast cell carcinogenesis by triclocarban and intervention by curcumin.

More than 85% of breast cancers are sporadic and attributable to long-term exposure to environmental carcinogens and co-carcinogens. To identify co-carcinogens with abilities to induce cellular pre-malignancy, we studied the activity of triclocarban (TCC), an antimicrobial agent commonly used in household and personal care products. Here, we demonstrated, for the first time, that chronic exposure to TCC at physiologically-achievable nanomolar concentrations resulted in progressive carcinogenesis of human breast cells from non-cancerous to pre-malignant. Pre-malignant carcinogenesis was measured by increasingly-acquired cancer-associated properties of reduced dependence on growth factors, anchorage-independent growth and increased cell proliferation, without acquisition of cellular tumorigenicity. Long-term TCC exposure also induced constitutive activation of the Erk-Nox pathway and increases of reactive oxygen species (ROS) in cells. A single TCC exposure induced transient induction of the Erk-Nox pathway, ROS elevation, increased cell proliferation, and DNA damage in not only non-cancerous breast cells but also breast cancer cells. Using these constitutively- and transiently-induced changes as endpoints, we revealed that non-cytotoxic curcumin was effective in intervention of TCC-induced cellular pre-malignancy. Our results lead us to suggest that the co-carcinogenic potential of TCC should be seriously considered in epidemiological studies to reveal the significance of TCC in the development of sporadic breast cancer. Using TCC-induced transient and constitutive endpoints as targets will likely help identify non-cytotoxic preventive agents, such as curcumin, effective in suppressing TCC-induced cellular pre-malignancy.

Synergistic induction of cancer cell death and reduction of clonogenic resistance by cisplatin and FK228.

Human urinary bladder cancer is the fifth most common cancer in the United States, and the long-term disease-free survival in patients is still suboptimal with current chemotherapeutic regimens. Development of effective chemotherapeutic regimens is crucial to decrease the morbidity and mortality of this cancer. The goal of this study was to investigate the effectiveness of FK228 in increasing cisplatin's ability to induce bladder cancer cell death and reduce drug resistance. Our study revealed that FK228 combined with cisplatin synergistically induced cell death and reduced clonogenic survival of human urinary bladder cancer cells. The Erk-Nox pathway played an important role in mediating signals highly increased by this combined treatment to induce significantly-elevated levels of reactive oxygen species, leading to substantially-induced caspase activation and synergistically-increased death in cancer cells. Cisplatin was able to enhance the ability of FK228 to significantly reduce glutathione, indicating a novel activity of combined FK228 and cisplatin in reducing drug resistance. The ability of combined FK228 and cisplatin to synergistically induce cell death and reduce clonogenic survival was also applicable to colon cancer cells. Hence, combined use of FK228 with cisplatin should be considered in development of therapeutic strategies to control urinary bladder cancer and other cancer development and recurrence.

Intervention of human breast cell carcinogenesis chronically induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.

More than 85% of breast cancers are sporadic and attributable to long-term exposure to environmental carcinogens, such as those in the diet, through a multistep disease process progressing from non-cancerous to premalignant and malignant stages. The chemical carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is one of the most abundant heterocyclic amines found in high-temperature cooked meats and is recognized as a mammary carcinogen. However, the PhIP's mechanism of action in breast cell carcinogenesis is not clear. Here, we demonstrated, for the first time, that cumulative exposures to PhIP at physiologically achievable, pico to nanomolar concentrations effectively induced progressive carcinogenesis of human breast epithelial MCF10A cells from a non-cancerous stage to premalignant and malignant stages in a dose- and exposure-dependent manner. Progressive carcinogenesis was measured by increasingly- acquired cancer-associated properties of reduced dependence on growth factors, anchorage-independent growth, acinar-conformational disruption, proliferation, migration, invasion, tumorigenicity with metastasis and increased stem-like cell populations. These biological changes were accompanied by biochemical and molecular changes, including upregulated H-Ras gene expression, extracellular signal-regulated kinase (ERK) pathway activation, Nox-1 expression, reactive oxygen species (ROS) elevation, increased HIF-1α, Sp1, tumor necrosis factor-α, matrix metalloproteinase (MMP)-2, MMP-9, aldehyde dehydrogenase activity and reduced E-cadherin. The Ras-ERK-Nox-ROS pathway played an important role in not only initiation but also maintenance of cellular carcinogenesis induced by PhIP. Using biological, biochemical and molecular changes as targeted endpoints, we identified that the green tea catechin components epicatechin-3-gallate and epigallocatechin-3-gallate, at non-cytotoxic doses, were capable of suppressing PhIP-induced cellular carcinogenesis and tumorigenicity.

Green tea catechin intervention of reactive oxygen species-mediated ERK pathway activation and chronically induced breast cell carcinogenesis.

Long-term exposure to low doses of environmental carcinogens contributes to sporadic human breast cancers. Epidemiologic and experimental studies indicate that green tea catechins (GTCs) may intervene with breast cancer development. We have been developing a chronically induced breast cell carcinogenesis model wherein we repeatedly expose non-cancerous, human breast epithelial MCF10A cells to bioachievable picomolar concentrations of environmental carcinogens, such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene (B[a]P), to progressively induce cellular acquisition of cancer-associated properties, as measurable end points. The model is then used as a target to identify non-cytotoxic preventive agents effective in suppression of cellular carcinogenesis. Here, we demonstrate, for the first time, a two-step strategy that initially used end points that were transiently induced by short-term exposure to NNK and B[a]P as targets to detect GTCs capable of blocking the acquisition of cancer-associated properties and subsequently used end points constantly induced by long-term exposure to carcinogens as targets to verify GTCs capable of suppressing carcinogenesis. We detected that short-term exposure to NNK and B[a]P resulted in elevation of reactive oxygen species (ROS), leading to Raf-independent extracellular signal-regulated kinase (ERK) pathway activation and subsequent induction of cell proliferation and DNA damage. These GTCs, at non-cytotoxic levels, were able to suppress chronically induced cellular carcinogenesis by blocking carcinogen-induced ROS elevation, ERK activation, cell proliferation and DNA damage in each exposure cycle. Our model may help accelerate the identification of preventive agents to intervene in carcinogenesis induced by long-term exposure to environmental carcinogens, thereby safely and effectively reducing the health risk of sporadic breast cancer.

Reactive oxygen species-mediated therapeutic control of bladder cancer.

Urinary bladder cancer is the fifth most common cancer in the US and the most costly cancer to manage because it requires life-long surveillance to monitor for recurrence and advanced progression. Urothelial carcinomas account for more than 90% of urinary bladder cancer cases. Transurethral resection and intravesical chemotherapy or immunotherapy are effective short-term treatments of urothelial carcinoma, but long-term management has not yet been optimized. Recent therapeutic strategies emphasize the targeted interference with aberrantly-regulated signaling modulators that result from genomic alterations. However, targeted therapeutic agents might not distinguish cancer cells from their normal counterparts, resulting in undesirable adverse effects. Thus, a new approach for the treatment of urothelial carcinoma has been suggested that differentially augments cancer-associated events, leading to selective death of cancer cells but not normal cells. Many aberrantly-regulated signaling modulators are associated with the elevation of reactive oxygen species (ROS), and an increasing number of studies report agents with the ability to induce ROS in cancer cells. Accordingly, therapeutic augmentation of ROS to a lethal level in cancer cells only would induce selective death of tumor cells but not normal cells, leading to a highly effective chemotherapy strategy for urothelial carcinoma.

Oncogenic H-Ras, FK228, and exogenous H2O2 cooperatively activated the ERK pathway in selective induction of human urinary bladder cancer J82 cell death.

More than 35% of human urinary bladder cancers involve oncogenic H-Ras activation. The goal of this study was to investigate the role of the ERK pathway in mediating apoptotic signals induced by oncogenic H-Ras, FK228 treatment, and exogenous H(2) O(2) treatment to increase Nox-1 elevation, leading to production of intracellular reactive oxygen species (ROS) for inducing apoptosis in human bladder cancer J82 cells. Our study revealed that FK228 combined with exogenous H(2)O(2) cooperatively induced activation of Mek1/2 and Erk1/2 to increase Nox-1 elevation, intracellular ROS production, caspase activation, and cell death. Expression of oncogenic H-Ras significantly increased these FK228- and exogenous H(2)O(2)-induced effects. Oncogenic H-Ras-increased cell susceptibility to FK228 could be alternately achieved by additional treatment with exogenous H(2)O(2). Hence, combined use of FK228 with ROS-generating agents may apply to therapeutic strategies to preferentially kill malignant cells with or without oncogenic H-Ras activation.

Differential induction of reactive oxygen species through Erk1/2 and Nox-1 by FK228 for selective apoptosis of oncogenic H-Ras-expressing human urinary bladder cancer J82 cells.

PURPOSE: This study sought to reveal mechanisms for differential regulation of reactive oxygen species (ROS) in histone deacetylase inhibitor FK228-induced selective apoptosis of oncogenic H-Ras-expressing human cancer cells. METHODS: Human urinary bladder cancer J82 and oncogenic H-Ras-expressing J82 cells were used to reveal FK228-induced differential Erk1/2 activation, Nox-1 elevation, ROS production, glutathione (GSH) depletion, caspase activation, and apoptosis. Specific inhibitors were used to suppress Nox-1 activity and ROS production. Mek1/2 inhibitor was used to suppress Erk1/2 activation. Validated-specific siRNAs were used to knock down Nox-1. ROS levels, GSH levels, and caspase-3/7 activities were measured by GSH assay, flow cytometry and luminescence assays, respectively. Western blot analysis determined levels of Erk1/2 and Nox-1. RESULTS: Erk1/2, Nox-1, ROS, caspase-3/7, and cell death were differentially induced, whereas GSH was differentially depleted by FK228 in oncogenic H-Ras-expressing J82 versus parental cells. Blockage of the ERK pathway resulted in suppressing oncogenic H-Ras- and FK228-induced Nox-1 elevation, ROS production, caspase activation, and cell death. Knockdown of Nox-1 by specific siRNAs reduced FK228-induced ROS production, caspase activation, and cell death. CONCLUSION: Oncogenic H-Ras expression and FK228 treatment synergistically induced the ERK pathway, resulting in differentially increased Nox-1 elevation, ROS production, and GSH depletion, leading to differential caspase activation and cell death in oncogenic H-Ras-expressing J82 versus parental cells.

FK228 and oncogenic H-Ras synergistically induce Mek1/2 and Nox-1 to generate reactive oxygen species for differential cell death.

To investigate the mechanism behind the pro-apoptotic ability of oncogenic H-Ras to enhance FK228-induced apoptosis, we primarily used the 10T1/2-TR-H-Ras cell line, in which ectopic expression of oncogenic H-Ras(V12) is controlled by the addition of tetracycline into cultures, and secondarily used oncogenic H-Ras-expressing MCF10A cells in our studies. Our results showed the pro-apoptotic roles of Mek1/2 activation, nicotinamide adenine dinucleotide phosphate-oxidase 1 (Nox-1) elevation, and reactive oxygen species (ROS) production in FK228-induced selective cell death of oncogenic H-Ras-expressing cells versus counterpart cells. We found that although Nox-1 elevation and ROS production played essential roles in oncogenic H-Ras-induced cell proliferation and morphological transformation, the expression of oncogenic H-Ras and FK228 treatment synergistically induced activation of Mek1/2. This activation resulted in differentially increased Nox-1 elevation and ROS production leading to selective cell death of oncogenic H-Ras-expressing cells versus counterpart cells. We also found that FK228 treatment induced mitochondrial ROS and Mek1/2 activation, bypassing Raf-1, to downstream Erk1/2, participating in the induction of selective cell death. Thus, the pro-apoptotic abilities of Mek1/2 and Nox-1 should be considered as potential targets in designing therapeutic protocols using FK228 to assure ROS-mediated cell death for treating cancer cells acquiring Ras activation.

Role of reactive oxygen species in proapoptotic ability of oncogenic H-Ras to increase human bladder cancer cell susceptibility to histone deacetylase inhibitor for caspase induction.

PURPOSE: Reveal mechanisms for the novel proapoptotic ability of oncogenic H-Ras to increase cell susceptibility to a histone deacetylase inhibitor (HDACI) FR901228 for inducing caspase activation and selective apoptosis. METHODS: Human urinary bladder cancer J82 and oncogenic H-Ras-expressing J82 cells were used to reveal differential induction of intracellular reactive oxygen species (ROS), caspase activation, and apoptosis by HDACI FR901228. ROS levels and caspase-8, -9, and -3/7 activities were measured by flow cytometry and luminescence assays, respectively. Specific inhibitors were used to suppress caspases and ROS. Western blot analysis determined modulators of caspase pathways. RESULTS: ROS, caspase activity, and cell death was differentially increased by FR901228 in oncogenic H-Ras-expressing J82 versus parental cells. Blocking ROS resulted in reduced FR901228-induced cell death and caspase activation. Suppression of caspase-8 resulted in reduced FR901228-activated caspase-9 and -3/7. Suppression of caspase-9 resulted in reduced FR901228-activated caspase-3/7. Although FR901228 induced an ROS-dependent increase of FasL, FasL failed to induce caspase activation and cell death. CONCLUSION: Increased ROS played an important role in the activation of the extrinsic and intrinsic caspase pathways to cooperatively induce executioner caspase-3/7 through a novel FasL-independent pathway in FR901228-induced selective apoptosis of oncogenic H-Ras-expressing J82 versus parental cells.

Precancerous model of human breast epithelial cells induced by NNK for prevention.

Epidemiological investigations have suggested that exposure to tobacco and environmental carcinogens increase the risk of developing human breast cancer. In light of the chronic exposure of human breast tissues to tobacco and environmental carcinogens, we have taken an approach of analyzing cellular changes of immortalized non-cancerous human breast epithelial MCF10A cells during the acquisition of cancerous properties induced by repeated exposure to the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) at a low concentration of 100 pM. We found that accumulated exposures of MCF10A cells to NNK result in progressive development of cellular carcinogenesis from a stage of immortalization to precancerous sub-stages of acquiring a reduced dependence on growth factors and acquiring anchorage-independent growth. Using Matrigel for MCF10A cells to form size-restricted acini, we detected that exposures to NNK resulted in altered acinar conformation. Analysis of gene expression profiles by cDNA microarrays revealed up- and down-regulated genes associated with NNK-induced carcinogenesis. Using this cellular carcinogenesis model as a target system to identify anticancer agents, we detected that grape seed proanthocyanadin extract significantly suppressed NNK-induced carcinogenesis of MCF10A cells. Our studies provide a carcinogenesis-cellular model mimicking the accumulative exposure to carcinogens in the progression of human breast epithelial cells to increasingly acquire cancerous properties, as likely occurs in the development of precancerous human breast cells. Our cellular model also serves as a cost-efficient, in vitro system to identify preventive agents that inhibit human breast cell carcinogenesis induced by chronic exposures to carcinogens.

Pro-apoptotic activity of oncogenic H-Ras for histone deacetylase inhibitor to induce apoptosis of human cancer HT29 cells.

PURPOSE: To verify the pro-apoptotic activity of oncogenic H-Ras in the increased susceptibility of human cancer cells to histone deacetylase inhibitor (HDACI). METHODS: The pro-apoptotic activity of oncogenic H-Ras(V12) was verified by its ability to increase susceptibility of human colorectal adenocarcinoma HT29 cells to HDACI for inducing apoptosis and growth inhibition, assayed by various methods. The mode of action of HDACI FR901228 was studied by its ability to modulate protein phosphorylation, acetylation, and expression levels in various signaling pathways, measured by Western blot analysis. RESULTS: Activation of caspase-3, -7, and -8, and serine protease by FR901228 was facilitated by oncogenic H-Ras to induce apoptosis. Expression of H-Ras(V12) changed the intrinsic modulation of Raf in cells responding to FR901228 treatment. Both p21( Cip1 ) and p27( Kip1 ) were induced in FR901228-treated cells arrested in either the G0/G1 or G2/M phase of the cell cycle. Deacetylation of FR901228-induced acetylation of core histones was accelerated by H-Ras(V12) in cells undergoing apoptosis. CONCLUSION: Expression of H-Ras(V12) increased susceptibility of HT29 cells to HDACI FR901228 and Trichostatin A for inducing apoptosis. The pro-apoptotic activity of H-Ras(V12) responding to HDACI indicates a potential value of this new class of anticancer agents in treating Ras-related human cancers.