Loss of Trefoil Factor 1 Accelerates the Immune Response to Colorectal Cancer.

BACKGROUND/AIM: Recent studies suggest that PD-L1 expression in immune cells, rather than tumor cells, plays a key role in tumor immunity. Trefoil factor family 1 (TFF1) is a secreted protein expressed mainly by the gastrointestinal epithelium and is related to the development of malignant disease. This study investigated the effects of TFF1 on tumor immunity in a xenograft mouse model of colorectal cancer (CRC). MATERIALS AND METHODS: MC38 cells were implanted in wild-type (WT) and TFF1KO mice, and the tumor micro-environment was investigated using immunohistochemistry. The circulating immune cells were analyzed using flow cytometry. RESULTS: Tumor growth was suppressed in TFF1KO mice. In the tumor microenvironment, CD8- and CD4-positive T cells and CD11c-positive dendritic cells (DCs) were frequently found in TFF1KO mice. When an immune checkpoint inhibitor was administered to these mice, almost half of the tumors in TFF1KO mice showed a complete response. The number of circulating PD-L1/DCs was markedly associated with tumor volume, with TFF1 deletion accelerating this effect and its injection decreasing it. These findings indicate that loss of TFF1 activates tumor immunity via frequent T-cell priming by DCs, and eventually suppresses tumor growth in CRC. In addition, the number of circulating PD-L1/DCs was identified as a predictive marker of checkpoint-inhibiting therapy efficacy. CONCLUSION: Loss of TFF1 resulted in accelerated immune response to colorectal cancer. Further studies are needed to investigate the precise mechanisms of TFF1 in immunotolerance and develop a novel TFF1-inhibiting immunotherapeutic strategy for CRC.

Dysfunction of infiltrating cytotoxic CD8+ T cells within the graft promotes murine kidney allotransplant tolerance.

Tolerance of mouse kidney allografts arises in grafts that develop regulatory tertiary lymphoid organs (rTLOs). Single-cell RNA-seq (scRNA-seq) data and adoptive transfer of alloreactive T cells after transplantation showed that cytotoxic CD8+ T cells are reprogrammed within the accepted graft to an exhausted/regulatory-like phenotype mediated by IFN-γ. Establishment of rTLOs was required because adoptive transfer of alloreactive T cells prior to transplantation results in kidney allograft rejection. Despite the presence of intragraft CD8+ cells with a regulatory phenotype, they were not essential for the induction and maintenance of kidney allograft tolerance since renal allotransplantation into CD8-KO recipients resulted in acceptance and not rejection. Analysis of scRNA-seq data from allograft kidneys and malignant tumors identified similar regulatory-like cell types within the T cell clusters and trajectory analysis showed that cytotoxic CD8+ T cells are reprogrammed into an exhausted/regulatory-like phenotype intratumorally. Induction of cytotoxic CD8+ T cell dysfunction of infiltrating cells appears to be a beneficial mechanistic pathway that protects the kidney allotransplant from rejection through a process we call "defensive tolerance." This pathway has implications for our understanding of allotransplant tolerance and tumor resistance to host immunity.

A clinical assessment of three-dimensional-printed liver model navigation for thrice or more repeated hepatectomy based on a conversation analysis.

PURPOSES: We performed a conversation analysis of the speech conducted among the surgical team during three-dimensional (3D)-printed liver model navigation for thrice or more repeated hepatectomy (TMRH). METHODS: Seventeen patients underwent 3D-printed liver navigation surgery for TMRH. After transcription of the utterances recorded during surgery, the transcribed utterances were coded by the utterer, utterance object, utterance content, sensor, and surgical process during conversation. We then analyzed the utterances and clarified the association between the surgical process and conversation through the intraoperative reference of the 3D-printed liver. RESULTS: In total, 130 conversations including 1648 segments were recorded. Utterance coding showed that the operator/assistant, 3D-printed liver/real liver, fact check (F)/plan check (Pc), visual check/tactile check, and confirmation of planned resection or preservation target (T)/confirmation of planned or ongoing resection line (L) accounted for 791/857, 885/763, 1148/500, 1208/440, and 1304/344 segments, respectively. The utterance's proportions of assistants, F, F of T on 3D-printed liver, F of T on real liver, and Pc of L on 3D-printed liver were significantly higher during non-expert surgeries than during expert surgeries. Confirming the surgical process with both 3D-printed liver and real liver and performing planning using a 3D-printed liver facilitates the safe implementation of TMRH, regardless of the surgeon's experience. CONCLUSIONS: The present study, using a unique conversation analysis, provided the first evidence for the clinical value of 3D-printed liver for TMRH for anatomical guidance of non-expert surgeons.

Liver remnant volume to body weight ratio of 0.65% as a lower limit in right hepatic trisectionectomy with bile duct resection.

BACKGROUND: Previous studies have suggested the utility of an indocyanine green plasma clearance rate of the future liver remnant (FLR) (ICGK-F) ≥0.05 in hepatobiliary resection to reduce the surgical risk. The present study aimed to verify whether future liver remnant size rather than ICGK-F matters in extended hepatobiliary resection. METHODS: Between 2004 and 2021, patients who underwent right hepatic trisectionectomy with bile duct resection were included. The effect of the FLR volume-to-body weight ratio (FLR/BW) and ICGK-F on posthepatectomy liver failure was evaluated along with other parameters. RESULTS: Among 91 study patients, the median ICGK-F, FLR, and FLR/BW were 0.057 (range, 0.027-0.099), 392 mL (145-705), and 0.78% (0.40-1.37), respectively. Posthepatectomy liver failure occurred in 23 patients. The incidence was 10 (40%) in 25 patients with an ICGK-F <0.05 and 12 (18%) in 65 patients with an ICGK-F ≥0.05 (P = .053); 13 (52%) in 25 patients with a FLR/BW <0.65% and 10 (15%) in 66 patients with a FLR/BW ≥0.65% (P = .001). Multivariate analysis showed that a FLR/BW <0.65% (odds ratio, 11.7; P = .005), age ≥65 years (odds ratio, 31.7; P < .001), and blood loss ≥25 mL/kg (odds ratio, 22.1; P = .004) were independent predictors of posthepatectomy liver failure, but ICGK-F <0.05 was not (P = .499). According to the meeting number of 3 factors, posthepatectomy liver failure incidence was 0 of 22 (0%) in patients with 0 factors, 6 of 43 (14%) in patients with 1, and 17 of 26 (65%) in patients with 2 or 3 (P < .001). CONCLUSION: A FLR/BW ≥0.65% may serve as a volumetric basis to reduce posthepatectomy liver failure after extended hepatobiliary resection.

The Goal of Intraoperative Blood Loss in Major Hepatobiliary Resection for Perihilar Cholangiocarcinoma: Saving Patients From a Heavy Complication Burden.

OBJECTIVE: To determine the goal of intraoperative blood loss in hepatectomy for perihilar cholangiocarcinoma. BACKGROUND: Although massive bleeding can negatively affect the postoperative course, the target value of intraoperative bleeding to reduce its adverse impact is unknown. METHODS: Patients who underwent major hepatectomy for perihilar cholangiocarcinoma between 2010 and 2019 were included. Intraoperative blood loss was adjusted for body weight [adjusted blood loss (aBL)], and the overall postoperative complications were evaluated by the comprehensive complication index (CCI). The impact of aBL on CCI was assessed by the restricted cubic spline regression. RESULTS: A total of 425 patients were included. The median aBL was 17.8 (interquartile range, 11.8-26.3) mL/kg, and the CCI was 40.6 (33.7-49.5). Sixty-three (14.8%) patients had an aBL<10 mL/kg, nearly half (45.4%) of the patients were in the range of 10 ≤aBL<20 mL/kg, and 37 (8.7%) patients had an aBL >40 mL/kg. The spline regression analysis showed a nonlinear incremental association between aBL and CCI; CCI remained flat with an aBL under 10 mL/kg; increased significantly with an aBL ranging from 10 to 20 mL/kg; grew gradually with an aBL over 20 mL/kg. These inflection points of ~10 and 20 mL/kg were almost consistent with the cutoff values identified by the recursive partitioning technique. After adjusting for other risk factors for the postoperative course, the spline regression identified a similar model. CONCLUSIONS: aBL had a nonlinear aggravating effect on CCI after hepatectomy for perihilar cholangiocarcinoma. The primary goal of aBL should be <10 mL/kg to minimize CCI.

Cyclodextrin Conjugated α-Bisabolol Suppresses FAK Phosphorylation and Induces Apoptosis in Pancreatic Cancer.

BACKGROUND/AIM: α-Bisabolol is an essential oil component extracted from plants, such as chamomile. We have previously reported that α-bisabolol suppressed proliferation, invasion, and motility of pancreas cancer. Cyclodextrin improved the solubility of α-bisabolol, therefore it enabled to administer intravenously. The aim of this study was to clarify the effect of cyclodextrin conjugated α-bisabolol (CD-BSB) and the signals pathways associated with α-bisabolol for pancreatic cancer. MATERIALS AND METHODS: Human pancreatic cancer cell lines were treated with or without CD-BSB. Cytomorphology and apoptosis were assessed in these treated groups. In addition, several phosphorylated proteins were analyzed to clarify the signal pathway concerning CD-BSB. In subcutaneous xenograft model, tumor volume and Ki-67 expression were evaluated among Control (untreated), CD-BSB, or Gemcitabine (GEM). RESULTS: CD-BSB significantly changed cytomorphology and induced apoptosis in pancreatic cancer cells. CD-BSB suppressed phosphorylation of focal adhesion kinase (FAK). In addition, pFAK 397 was inhibited by CD-BSB in a concentration-dependent manner in cancer cells. In the subcutaneous xenograft models, the tumor volume in the CD-BSB groups was lower than Control groups. Ki67-positive cells in CD-BSB treated group were lower than the GEM-treated groups. CONCLUSION: We clarified the efficiency of CD-BSB in xenograft tumor using intravenous administration. α-Bisabolol suppresses phosphorylation of FAK 397 and impairs cytoskeletal polymerization in a pancreatic cancer cell line. Further investigations are required to reveal the precise mechanisms of the antitumor effects of solubilized α-bisabolol to facilitate its clinical application. Our data indicate that solubilized α-bisabolol has therapeutic potential and could improve the prognosis of cancer patients.

"Evolving Trends in Pancreatic Cystic Tumors: A 3-Decade Single-Center Experience With 1290 Resections".

OBJECTIVE: The aim of this study was to describe our institutional experience with resected cystic tumors of the pancreas with emphasis on changes in clinical presentation and accuracy of preoperative diagnosis. SUMMARY BACKGROUND DATA: Incidental discovery of pancreatic cystic lesions has increased and has led to a rise in pancreatic resections. It is important to analyze surgical outcomes from these procedures, and the prevalence of malignancy, pre-malignancy and resections for purely benign lesions, some of which may be unintended. METHODS: Retrospective review of a prospective database spanning 3 decades. Presence of symptoms, incidental discovery, diagnostic studies, type of surgery, postoperative outcomes, and concordance between presumptive diagnosis and final histopathology were recorded. RESULTS: A total of 1290 patients were identified, 62% female with mean age of 60 years. Fifty-seven percent of tumors were incidentally discovered. Ninety-day operative mortality was 0.9% and major morbidity 14.4%. There were 23 different diagnosis, but IPMN, MCN, and serous cystadenoma comprised 80% of cases. Concordance between preoperative and final histopathological diagnosis increased by decade from 45%, to 68%, and is presently 80%, rising in parallel with the use of endoscopic ultrasound, cytology, and molecular analysis. The addition of molecular analysis improved accuracy to 91%. Of misdiagnosed cases, half were purely benign and taken to surgery with the presumption of malignancy or premalignancy. The majority of these were serous cystadenomas. CONCLUSIONS: Indications and diagnostic work-up of cystic tumors of the pancreas have changed over time. Surgical resection can be performed with very low mortality and acceptable morbidity and diagnostic accuracy is presently 80%. About 10% of patients are still undergoing surgery for purely benign lesions that were presumed to be malignant or premalignant. Further refinements in diagnostic tests are required to improve accuracy.

Identification of cell cycle-associated and -unassociated regulators for expression of a hepatocellular carcinoma oncogene cyclin-dependent kinase inhibitor 3.

Human cyclin-dependent kinase inhibitor 3 (CDKN3) is a known oncogene in hepatocellular carcinoma (HCC) and its expression is promoted during tumor development. CDKN3 serves as a cell cycle regulator and its dysregulation is considered to be a causal factor for tumor progression. However, the molecular basis of the regulation of CDKN3 expression remains largely elusive. Using in silico approach, we identified CDKN3SE, a super enhancer (SE), and enhancer RNA (eRNA) candidates transcribed from this SE. Among the eRNA candidates, the expression of CDKN3eRNA was detected in the human HCC model cell line HepG2, and was found to facilitate the expression of CDKN3 without affecting the cell proliferation rate. In silico screening revealed two DNA-binding transcription factors, upstream stimulatory factor (USF) 1 and 2, involved in the regulation of CDKN3eRNA expression on CDKN3SE. A knock-down of USF1/USF2 expression in the HepG2 cells did not affect CDKN3eRNA expression, while the expression of CDKN3 was down-regulated. In a USF2 dominant negative HepG2 cell line generated by genome editing, a drastically altered cell shape and lowered cell proliferation rate were found; however, the expression of CDKN3eRNA appeared unaffected. Thus, the present study illustrated two regulators for CDKN3 expression: USF2, as a cell cycle-associated protein regulator, and CDKN3eRNA, as a cell cycle-unassociated RNA regulator.

Prediction of R Status in Resections for Pancreatic Cancer Using Simplified Radiological Criteria.

OBJECTIVE: Predicting R status before surgery for pancreatic cancer (PDAC) patients with upfront surgery and neoadjuvant therapy. SUMMARY BACKGROUND DATA: Negative surgical margins (R0) are a key predictor of long-term outcomes in PDAC. METHODS: Patients undergoing pancreatic resection with curative intent for PDAC were identified. Using the CT scans from the time of diagnosis, the 2019 NCCN borderline resectability criteria were compared to novel criteria: presence of any alteration of the superior mesenteric-portal vein (SMPV) and perivascular stranding of the superior mesenteric artery (SMA). Accuracy of predicting R status was evaluated for both criteria. Patient baseline characteristics, surgical, histopathological parameters, and long-term overall survival (OS) after resection were evaluated. RESULTS: A total of 593 patients undergoing pancreatic resections for PDAC between 2010 and 2018 were identified. Three hundred and twenty-five (54.8%) patients underwent upfront surgery, whereas 268 (45.2%) received neoadjuvant therapy. In upfront resected patients, positive SMA stranding was associated with 56% margin positive resection rates, whereas positive SMA stranding and SMPV alterations together showed a margin positive resection rate of 75%. In contrast to these criteria, the 2019 NCCN borderline criteria failed to predict margin status. In patients undergoing neoadjuvant therapy, only perivascular SMA stranding remained a predictor of margin positive resection, leading to a rate of 33% R+ resections. Perivascular SMA stranding was related to higher clinical T stage (P = 0.003) and clinical N stage (P = 0.043) as well as perineural invasion (P = 0.022). SMA stranding was associated with worse survival in both patients undergoing upfront surgery (36 vs 22 months, P = 0.002) and neoadjuvant therapy (47 vs 34 months, P = 0.050). CONCLUSIONS: The novel criteria were accurate predictors of R status in PDAC patients undergoing upfront resection. After neoadjuvant treatment, likelihood of positive resection margins is approximately halved, and only perivascular SMA stranding remained a predictive factor.

Concepts and Outcomes of Perioperative Therapy in Stage IA-III Pancreatic Cancer-A Cross-Validation of the National Cancer Database (NCDB) and the German Cancer Registry Group of the Society of German Tumor Centers (GCRG/ADT).

(1) Background: The aim of this study is to assess perioperative therapy in stage IA-III pancreatic cancer cross-validating the German Cancer Registry Group of the Society of German Tumor Centers-Network for Care, Quality, and Research in Oncology, Berlin (GCRG/ADT) and the National Cancer Database (NCDB). (2) Methods: Patients with clinical stage IA-III PDAC undergoing surgery alone (OP), neoadjuvant therapy (TX) + surgery (neo + OP), surgery+adjuvantTX (OP + adj) and neoadjuvantTX + surgery + adjuvantTX (neo + OP + adj) were identified. Baseline characteristics, histopathological parameters, and overall survival (OS) were evaluated. (3) Results: 1392 patients from the GCRG/ADT and 29,081 patients from the NCDB were included. Patient selection and strategies of perioperative therapy remained consistent across the registries for stage IA-III pancreatic cancer. Combined neo + OP + adj was associated with prolonged OS as compared to neo + OP alone (17.8 m vs. 21.3 m, p = 0.012) across all stages in the GCRG/ADT registry. Similarly, OS with neo + OP + adj was improved as compared to neo + OP in the NCDB registry (26.4 m vs. 35.4 m, p < 0.001). (4) Conclusion: The cross-validation study demonstrated similar concepts and patient selection criteria of perioperative therapy across clinical stages of PDAC. Neoadjuvant therapy combined with adjuvant therapy is associated with improved overall survival as compared to either therapy alone.

ABO blood group distribution and risk of malignancy in patients undergoing resection for intraductal papillary mucinous neoplasm (IPMN).

BACKGROUND: The ABO blood type has been associated with risk of development of several malignancies, including pancreatic cancer. Data regarding IPMN is equivocal. To investigate this further, we analyzed the association between the ABO blood group and the presence of malignancy in a large cohort of resected IPMN and its influence in survival. METHODS: 819 patients who underwent pancreatic resection for IPMN in the Massachusetts General Hospital (MGH) and Cambridge University Hospitals NHS Foundation Trust (CUH) from January 1993 to December 2020 were identified from prospective institutional databases. Pathological characteristics and blood type were correlated. RESULTS: The distribution of blood types A, B, AB and O was 384 (47%), 92 (11%), 44 (5%) and 299 (37%), respectively. This blood type distribution was different than the reference population of the MGH and the CUH, which is 55% non-O blood group, and 45% type O. There was a significant predominance of non-O blood types when compared with O-blood type in patients with malignant IPMN (i.e. patients with high-grade dysplasia and invasive cancer) (67% vs 33%, OR 1.31 95%CI: 0.98-1.75, p = 0.069). The association was stronger for IPMN with invasive cancer (OR 1.43 95%CI: 1.01-2.02, p = 0.039). Blood group did not influence survival. CONCLUSION: Non-O blood type is associated with need for resection in IPMN and with presence of invasive carcinoma.

T cells armed with C-X-C chemokine receptor type 6 enhance adoptive cell therapy for pancreatic tumours.

The efficacy of adoptive cell therapy for solid tumours is hampered by the poor accumulation of the transferred T cells in tumour tissue. Here, we show that forced expression of C-X-C chemokine receptor type 6 (whose ligand is highly expressed by human and murine pancreatic cancer cells and tumour-infiltrating immune cells) in antigen-specific T cells enhanced the recognition and lysis of pancreatic cancer cells and the efficacy of adoptive cell therapy for pancreatic cancer. In mice with subcutaneous pancreatic tumours treated with T cells with either a transgenic T-cell receptor or a murine chimeric antigen receptor targeting the tumour-associated antigen epithelial cell adhesion molecule, and in mice with orthotopic pancreatic tumours or patient-derived xenografts treated with T cells expressing a chimeric antigen receptor targeting mesothelin, the T cells exhibited enhanced intratumoral accumulation, exerted sustained anti-tumoral activity and prolonged animal survival only when co-expressing C-X-C chemokine receptor type 6. Arming tumour-specific T cells with tumour-specific chemokine receptors may represent a promising strategy for the realization of adoptive cell therapy for solid tumours.

Pre-exposure to Fluorouracil Increased Trifluridine Incorporation and Enhanced its Anti-tumor Effect for Colorectal Cancer.

BACKGROUND/AIM: Trifluridine/tipiracil (FTD/TPI) is used for metastatic colorectal cancer, that is refractory to 5-fluorouracil (5-FU)-based therapies. However, the impact of pre-exposure to 5-FU on the anti-cancer effect of FTD, which is a key component of FTD/TPI, is unclear. MATERIALS AND METHODS: The incorporation into DNA and anti-cancer activity of FTD were analyzed in several cancer cell lines under response to FTD treatment with or without 5-FU pre-exposure. The volumes of tumors in xenografted nude mice were examined among groups that were either untreated or treated with S-1, FTD/TPI or FTD/TPI with pre-exposure to S-1. RESULTS: Pre-exposure to 5-FU significantly increased FTD incorporation into DNA and enhanced its anti-cancer effect for viability and proliferation of cancer cells. In the xenograft nude mouse model, the tumor volumes in the FTD/TPI-treated and S-1-pre-exposed group were lower than those in the FTD/TPI-only-treated group. Although both FTD dose and exposure time in the FTD/TPI-treated and S-1-pre-exposed mice were smaller than those in the FTD/TPI-only-treated mice, the incorporated FTD in the tumors in the former group was 86.5% of that in the latter group. CONCLUSION: Pre-exposure to 5-FU enhanced the incorporation into DNA and the anti-cancer effect of FTD in the context of colorectal cancer. Our data indicate the potential for a new sequential therapy using S-1 and FTD/TPI to improve prognosis of colorectal cancer.

Establishment of consomic strains derived from A/J and SM/J mice for genetic analysis of complex traits.

Consomic strains, in which one chromosome is derived from a donor strain and the other chromosomes are derived from the recipient strain, provide a powerful tool for the dissection of complex genetic traits. In this study we established ten consomic strains (A-2(SM), A-6(SM), A-11(SM), A-12(SM), A-13(SM), A-15(SM), A-17(SM), A-18(SM), A-19(SM), A-Y(SM)) using the SM/J strain as the donor and the A/J strain as the recipient; these are the parental strains of a set of SMXA recombinant inbred (RI) strains that we had developed previously. We analyzed body weights and blood lipid levels in the consomic and parental strains. The mean values for each trait showed a continuous range of variation in the consomic strains suggesting that they are controlled by multiple genes. We previously identified suggestive QTLs for body weight on chromosome 6 in SMXA RI strains and (SM/J × A/J)F(2) mice. The observation that the A-6(SM) consomic strain had a significantly lower mean body weight than the A/J strain supports the presence of this QTL on chromosome 6. Similarly, the higher blood triglyceride level in the A-11(SM) strain shows the existence of a previously mapped QTL on chromosome 11, and the A-12(SM) strain provides evidence of a QTL for blood total cholesterol level on chromosome 12. These consomic strains, along with the previously developed set of SMXA RI strains from A/J and SM/J mice, offer an invaluable and powerful resource for the analysis of complex genetic traits in mice.

X-radiation-induced down-regulation of the EGF receptor in primary cultured rat hepatocytes.

Exposure to X radiation is associated with a decline in the proliferative activity of the liver, but the molecular mechanism(s) is not well understood. We investigated whether exposure to X radiation is involved in functional changes in the epidermal growth factor (EGF) receptor (EGFR), thereby causing a reduction of EGF-induced DNA synthesis using periportal hepatocytes (PPH) and perivenous hepatocytes (PVH), which differ in their proliferative activity. X radiation dose-dependently decreased DNA synthesis in both subpopulations. The rate of decline in the DNA synthesis was greater in PPH than in PVH, but the zonal difference disappeared after exposure to 10 Gy X radiation. [(125)I]EGF binding studies indicated that high-affinity EGFRs in both subpopulations were down-regulated after X irradiation. Furthermore, EGF-induced EGFR dimerization and phosphorylation at Y1173 in both subpopulations were down-regulated after X irradiation, and the rate of decline was greater in PPH than in PVH. In contrast, phosphorylation at Y845 after EGF treatment was dose-dependently up-regulated after X irradiation in both subpopulations. These results suggest that the X-radiation-related decline in EGF-induced DNA synthesis is caused at least partly by the modification of EGFR function.

Clinically relevant radioresistant cells efficiently repair DNA double-strand breaks induced by X-rays.

Radiotherapy is one of the major therapeutic modalities for eradicating malignant tumors. However, the existence of radioresistant cells remains one of the most critical obstacles in radiotherapy and radiochemotherapy. Standard radiotherapy for tumor treatment consists of approximately 2 Gy once a day, 5 days a week, over a period of 5-8 weeks. To understand the characteristics of radioresistant cells and to develop more effective radiotherapy, we established a novel radioresistant cell line, HepG2-8960-R with clinical relevance from parental HepG2 cells by long-term fractionated exposure to 2 Gy of X-rays. HepG2-8960-R cells continued to proliferate with daily exposure to 2 Gy X-rays for more than 30 days, while all parental HepG2 cells ceased. After exposure to fractionated 2 Gy X-rays, induction frequencies of micronuclei and remaining foci of gamma-H2AX in HepG2-8960-R were less than those in HepG2. Flow cytometric analysis revealed that the proportion of cells in S- and G2/M-phase of the cell cycle was higher in HepG2-8960-R than in HepG2. These suggest that the response of clinically relevant radioresistant (CRR) cells to fractionated radiation is not merely an accumulated response to each fractionated radiation. This is the first report on the establishment of a CRR cell line from an isogenic parental cell line.

Analysis of gene and protein expression of cytochrome P450 and stress-associated molecules in rat liver after spaceflight.

As the incidence and length of spaceflight increases, the need to administer pharmacological agents to crew members may become greater. The final goal is to predict the therapeutic or toxic effects of drugs, especially those with a low therapeutic index, during spaceflight. The liver is central to systemic metabolism, therefore, various drugs rely on hepatic metabolism by cytochrome P450 (CYP). The function of individual CYP enzymes is diverse because they are subject to different regulatory mechanisms and substrate specificity. Because spaceflight may be a stressor and influence each CYP expression individually, the purpose of the present study was to measure the expression of 11 CYP genes and protein distribution in the liver of rats after spaceflight, using real-time polymerase chain reaction and immunohistochemistry, respectively. The gene and protein expression of stress-related proteins such as cold-inducible RNA binding protein (Cirp), heat shock protein 70 (HSP70), HSP90 and the p53 tumor suppressor gene, were also determined. CYP4A1and Cirp gene expression was significantly increased whereas HSP90 and p53 gene expression was significantly decreased in the flight group than in the ground control. Combined with histology, it is concluded that the effects of spaceflight on the liver may be similar to mild cold stress or fasting.

Induction of apoptosis on K562 cell line and double strand breaks on colon cancer cell line expressing high affinity receptor for granulocyte macrophage-colony stimulating factor (GM-CSF).

BACKGROUND: Immunotoxins are comprised of both the cell targeting and the cell killing moieties. We previously established a new immunotoxin, i.e. Shiga toxin granulocyte macrophage-colony stimulating factor (StxA1-GM-CSF), comprises of catalytic domain of Stx, as a killing moiety and GM-CSF, as a cell targeting moiety. In this study, the ability of the immunotoxin to induce apoptosis and double strand breaks (DSB) on different cell lines was investigated. METHODS: The recombinant hybrid protein was expressed in bacterial expression system and purified with nickel-nitrilotriacetate acid resin. The K562 (erythroid leukemia) cell line and LS174 (colon carcinoma) were used in this study. The neutral comet assay was carried out for the detection of DSB and Hoechst staining was performed for apoptosis. RESULTS: StxA1-GM-CSF effectively induced apoptosis on K562 cell line and DNA Double Strand Break (DSB) were observed on colon cancer cell line treated with StxA1-GM-CSF. CONCLUSION: This novel action i.e. DNA damage might be a relevant mechanism of action for StxA1-GM-CSF that is designed to act as immunotoxin, although further investigation is required.

Gene expression profiles in mouse liver cells after exposure to different types of radiation.

The liver is one of the target organs of radiation-induced cancers by internal exposures. In order to elucidate radiation-induced liver cancers including Thorotrast, we present a new approach to investigate in vivo effects of internal exposure to alpha-particles. Adopting boron neutron capture, we separately irradiated Kupffer cells and endothelial cells in mouse liver in vivo and analyzed the changes in gene transcriptions by an oligonucleotide microarray. Differential expression was defined as more than 3-fold for up-regulation and less than 1/3 for under-regulation, compared with non-irradiated controls. Of 6,050 genes examined, 68 showed differential expression compared with non-irradiated mice. Real-time polymerase chain reaction validated the results of the microarray analysis. Exposure to alpha-particles and gamma-rays produced different patterns of altered gene expression. Gene expression profiles revealed that the liver was in an inflammatory state characterized by up-regulation of positive acute phase protein genes, irrespective of the target cells exposed to radiation. In comparison with chemical and biological hepatotoxicants, inductions of Metallothionein 1 and Hemopexin, and suppressions of cytochrome P450s are characteristic of radiation exposure. Anti-inflammatory treatment could be helpful for the prevention and protection of radiation-induced hepatic injury.