ADENOVIRUS-MEDIATED P53 GENE TRANSFER INCREASES THE THERMOSENSITIVITY OF HUMAN GASTRIC CARCINOMA CELL LINES(IN VITRO AND IN VIVO)

Objective: To evaluate the effect of adenovirus-mediated p53 gene (Adp53) on apoptosis andradiosensitivity of human gastric carcinoma cell lines.Methods: Recombinant adenovirus expressing wild-type p53 gene was transferred into four human gastric carcinoma cell lines with different p53 genetic status. p53 proteinexpression was detected by immunohistochemistry assayand western blot assay. Cell survival was assessed using a clonogenic assay. TUNEL assay was used in determination of apoptosis. Four human gastric carcinoma cells infectedwith Adp53 were irradiated with 4Gy and cell cycle distribution and Sub-G1 peak were assayed by flowcytometry. Results: G2/M arrest, apoptosis and inhibition of tumor cell proliferation were induced by infection atAdp53 at 100 MOI which caused high transfer rate ofwild-type p53 and strong expression of p53 protein in four human gastric carcinoma cells. The radio-enhancement ratio of Adp53 at 4Gy were 3.0 for W cell, 3.6 for M cell, 2.2 for neo cell and 2.5 for 823 cell in vitro. Conclusion: Thisstudy demonstrated that Adp53 transfer increased cellularapoptosis and radiosensitivity of human gastric carcinoma cell lines in vitro independently on cellular intrinsic p53status thus supporting the combination of p53 gene therapy with radiotherapy in clinical trials.

Adenovirus-mediated corneal endotheliitis: a case report

Dear Editor,Cornealendotheliitisisacommonandintriguing clinicalentitycharacterizedbycornealedema,keratic precipitates,and mild to moderate anterior chamber reaction,which occupies the important pathogenic factor of corneal blindness[1].Robin et al[2]first described a patient who suffered from intraocular inflammation and progressive corneal endotheliitis associated with herpes simplex infection.Since then,accumulating clinical evidence confirmed that its etiology was mainly attributed to the family of herpesviridae,including herpes simplex virus,varicella zoster virus,and cytomegalovirus,which initiated the direct cell damage and immune-and inflammatory-mediated lesion on endothelial cells[1,3].

Stereotaxic microinjection of adenovirus-mediated human tissue Kallikrein gene reduces apoptosis in a rat model of middle cerebral artery occlusion

BACKGROUND：Several studies have demonstrated that Kallikrein gene transfer provides neuroprotection. Whether the neuroprotective effects of human tissue Kallikrein （HTK） are associated with apoptosis remains unclear. OBJECTIVE： To investigate the effects of HTK on apoptosis in the peripheral cerebral infarct region. DESIGN, TIME AND SETTING： The completely randomized grouping, gene engineered, controlled experiment was performed at the Lin Baixin Laboratory Center, the Second Affiliated Hospital of Sun Yat-sun University between September 2007 and April 2008. MATERIALS： Ninety clean, healthy, male, Sprague Dawley rats were included. pUC19-HTK plasmid was constructed in the Laboratory for Neurology, the Second Affiliated Hospital of Sun Yat-sen University, China. bcl-2, bax, caspase-3, and β-actin were designed and purified by Shanghai Shuiyuan Company, China. METHODS： Middle cerebral artery occlusion （MCAO） model was established in all rats. At 72 hours after MCAO model establishment, rats were randomly divided into 3 groups, with 30 rats per group： blank control, saline, and pAdCMV-HTK. The saline and pAdCMV-HTK groups were respectively stereotactically micro-injected with 5 μL physiological saline or pAdCMV-HTK at the area surrounding the cerebral infarction region. Only puncture was performed, without any injection, in the blank control group. MAIN OUTCOME MEASURES： At 72 hours after MCAO establishment, as well as at 24 hours, 72 hours, and 7 days subsequent to treatment, exogenous HTK expression was detected by immunohistochemistry. Apoptosis was examined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling （TUNEL）, while mRNA levels of bcl-2, bax, and caspase-3 were detected by reverse transcription-polymerase chain reaction. In addition, neurological severity scores were evaluated prior to and after treatments. RESULTS： Ninety rats were included in the final analysis. HTK was primarily detected in the cytoplasm at 24 hours after pAdCMV-HTK injection. Thereafter, HTK expression gradually increased and reached a peak level at 72 hours after injection, which was significantly different from the blank control and saline groups （P 〈 0.05）. At 7 days, HTK expression began to decrease, but remained higher than the saline and blank control groups （P 〈 0.05）. Apoptotic cells aggregated around the cerebral infarction region. Compared with the saline and blank control groups, the mean number of TUNEL-positive cells was notably decreased in the pAdCMV-HTK group at each time point after treatment （P 〈 0.05）. mRNA levels of bcl-2, bax, and caspase-3 were elevated in all groups at 24 hours after treatment, peaked at 72 hours, and then gradually decreased again at 7 days. Compared with the saline and blank control groups, bcl-2 slightly increased, but was not significantly different from the pAdCMV-HTK group （P 〉 0.05）. bax and caspase-3 mRNA levels were significantly reduced at 24 and 72 hours after treatment （P 〈 0.05）. At 72 hours and, in particular, at 7 days after treatment, neurological severity scores were significantly less in the pAdCMV-HTK group compared with the saline and blank control groups （P 〈 0.05–0.01）. CONCLUSION： HTK could protect neural cells in the peripheral cerebral infarction region from apoptosis, which resulted in a better outcome. This may be related to modulated bcl-2 expression and reduced bax and caspase-3 expression.

Editor's Choice—Adenovirus-mediated gene transfection of stem cells

Neural stem cells transplantation plays an important role in repair and cell replacement therapy for nervous system degenerative diseases. However, the self-repair effects are minimal because of a limited absolute number, as well as the local microenvironment, post-transplantation. A combined treatment utilizing stem cell transplantation and gone therapy can exert a dual effect involving stem cells and neurotrophic factors. The adenovirus carrier is

Synergistic anticancer effect of exogenous wild-type p53 gene combined with 5-FU in human colon cancer resistant to 5-FU in vivo

AIM To investigate the anticancer effect of a recombinant adenovirus-mediated p53(r Ad-p53) combined with 5-fluorouracil(5-FU) in human colon cancer resistant to 5-FU in vivo and the mechanism of r Ad-p53 in reversal of 5-FU resistance.METHODS nude mice bearing human colon cancer SW480/5-FU(5-FU resistant) were randomly assigned to four groups(n = 25 each): control group, 5-FU group, r Ad-p53 group, and r Ad-p53 + 5-FU group. At 24 h, 48 h, 72 h, 120 h and 168 h after treatment, 5 mice were randomly selected from each group and sacrificed using an overdose of anesthetics. The tumors were removed and the protein expressions of p53, protein kinase C(PKC), permeability-glycoprotein(P-gp) and multidrug resistance-associated protein 1(MRP1)(Western blot) and apoptosis(TUNEL) were determined.RESULTS The area ratios of tumor cell apoptosis were larger in the r Ad/p53 + 5-FU group than that in the control, 5-FU and r Ad/p53 groups(P < 0.05), and were larger in the r Ad/p53 group than that of the control group(P < 0.05) and the 5-FU group at more than 48 h(P < 0.05). The p53 expression was higher in the r Ad/p53 and the r Ad/p53 + 5-FU groups than that of the control and 5-FU groups(P < 0.05), and were higher in the r Ad/p53 + 5-FU group than that of the r Ad/p53 group(P < 0.05). Overexpression of PKC, P-gp and MRP1 was observed in the 5-FU and control groups. In the r Ad/p53 + 5-FU group, the expression of P-gp and MRP1 was lower that of the control and 5-FU groups(P < 0.05), and the expression of PKC was lower than that of the control, 5-FU and r Ad/p53 groups at more than 48 h(P < 0.05). In the r Ad/p53 group, the expression of P-gp and MRP1 was lower that of the control and 5-FU groups at more than 48 h(P < 0.05), and the expression of PKC was lower than that of the control and 5-FU groups at more than 120 h(P < 0.05).CONCLUSION5-FU combined with r Ad-p53 has a synergistic anticancer effect in SW480/5-FU(5-FU resistance), which contributes to reversal of 5-FU resistance.