Subcellular localization of APOBEC3G by confocal laser scanning microscope (CLSM) / 病毒学报

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G) cDNA was amplified from total RNA prepared from nonpermissive H9 cells by RT-PCR. APOBEC3G cDNA is 1155nt long, encoding 384 amino acids. The APOBEC3G gene was then cloned into the eukaryotic expression vector pEGFP-C3. The generated pEGFP-3G construct was then transfected into CD4+ HeLa cell to determine the expression and the subcellular localization of GFP-APOBEC3G fusion protein. Under CLSM the localization of the expressed GFP-APOBEC3G in the cytoplasm of CD4+ HeLa cells was observed.

Therapeutic effect of AdCMVCD/5-FC system and metabolism of 5-FC in the treatment of human tongue squamous cell carcinoma / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the therapeutic effect and metabolism of 5-fluorocytosine (5-FC) in human tongue squamous carcinoma cells after treatment with adenovirus-mediated cytosine deaminase (AdCMVCD)/5-FC system.</p><p><b>METHODS</b>Human tongue squamous carcinoma cells (Tca8113 cell line) and its xenografts in BALB/c nude mice were treated with AdCMVCD/5-FC system. The killing effect in vitro and bystander effect were detected by microculture tetrazolium (MTT) assay. Tumor inhibition effect and histopathological changes were observed in vivo. High-performance liquid chromatography (HPLC) was performed to determine the metabolism of 5-FC in vitro and in vivo.</p><p><b>RESULTS</b>AdCMVCD/5-FC system had strong killing effect and bystander effect on Tca8113 cells. Both condition media and cell extracts showed two peaks identified as 5-FC and 5-fluorouracil (5-FU) by HPLC and a time-dependent generation of 5-FU and concomitant time-dependent decreases of 5-FC. Compared to the control groups, mice treated with AdCMVCD/5-FC system demonstrated significant tumor regression (P < 0.001); the tumor doubling time prolonged and inhibition rate was 92.62%. There were substantial tumor necrotic areas and infiltrative lymphocytes around necrotic areas in the AdCMVCD/5-FC treated group under light microscope. There was a significantly low concentration of 5-FC and high concentration of 5-FU in tumor tissue, but only 5-FC was found in blood.</p><p><b>CONCLUSION</b>AdCMVCD/5-FC suicide gene system had significant in vitro and in vivo anti-tumor effect on human tongue squamous cell carcinoma due to convert 5-FC into 5-FU.</p>

Three-pronged approach to treat rectal cancer: liposome transfer cytosine deaminase gene, ionizing radiation and dendritic cells / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the anticancer activity of three-pronged approach: liposome transfer cytosine deaminase (CD) gene, ionizing radiation, and dendritic cells (DCs).</p><p><b>METHODS</b>After rectal cancer cells were killed by CD and 5-FC and radiotherapy with improved liposome transfection and radiosensitization, the killed cancer cells, DCs and T lymphocytes were incubated together. The uptake of (3)H-TdR weighed the immune activity of DCs. The effect of anticancer was monitored by MTT.</p><p><b>RESULTS</b>DCs acquired the apoptosis body and other antigen from the killed cancer cells and presented them to T-lymphocytes. The induced CTL killed the cancer cells powerfully and selectively. The mortality of rectal cancer cells was 98.1%, 76.2%, 37.8% at the ratio of efficacy to target of 30:1, 15:1, 1:1, respectively.</p><p><b>CONCLUSION</b>The three-pronged approach may represent a powerful and safe means to selectively destroy cancer cells.</p>

Adenoviral mediated suicide gene transfer in the treatment of pancreatic cancer / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To determine the efficacy of adenovirus mediated suicide gene transduction combined with prodrug 5-fluorocytosine (5FC) as a therapeutic protocol for pancreatic cancer.</p><p><b>METHODS</b>Cytosine Deaminase(CD) gene was cloned into pAdTrack-CMV-CD, pAdTrack-CMV-CD and pAdEasy-1 were recombined in bacteria. The newly recombined adenovirus (Ad)-CD containing green fluorescent protein (GFP) were packaged and propagated in 293 cells and purified by cesium chloride gradient centrifugation. Human pancreatic carcinoma cell line-Patu8988 was infected with this virus, then 5FC was added. XTT assay was used to estimate relative numbers of viable cells. In vivo model of pancreatic cancer was established by injecting 1.0 x 10(7) Patu8988 cells subcutaneously in Balb/c nude mice. When tumors were palpable, Ad-CD was injected into each tumor and 5FC was administered.</p><p><b>RESULTS</b>Positive clones were selected using endonuclease to digest the recombinants and the concentration of viral liquids containing the CD gene was 2 x 10(11) pfu /ml. Significant cytotoxic activity as shown for 5FC in the CD gene transduced 8988 cell line, while little effect was found in the nontransduced pancreatic carcinoma cells. Antitumor effect was observed in Patu8988 xenograft nude mice with in situ CD gene transduction.</p><p><b>CONCLUSIONS</b>CD gene mediated by adenovirus has high infectivity and may be useful for gene therapy in pancreatic carcinoma. These data demonstrate the use of an enzyme prodrug strategy in experimental pancreatic cancer.</p>

X-ray combined with cytosine deaminase suicide gene therapy enhances killing of colorectal carcinoma cells in vitro / 中华外科杂志

<p><b>OBJECTIVE</b>To study the effect of X-ray on gene transfer and the antitumoral effect of X-ray combined with suicide gene therapy on colorectal carcinoma cells.</p><p><b>METHODS</b>Green fluorescent protein (GFP) was seen under fluorescent microscope. GFP gene was used for reporting gene to learn gene transfer efficiency and gene expressing time under the influence of radiation. G418 was used to select cytosine deaminase (CD) positive neoplasm cells and CD gene transfer efficiency was tested by cloning efficiency. Antitumoral effect of X-ray combined with CD and 5-FC on colorectal carcinoma cells was tested by MTT.</p><p><b>RESULTS</b>4 Gy radiation could improve supercoiled plasmid DNA transfer efficiency for about 2 - 4 times and 30 times for linearized plasmid DNA. The mean durations of GFP gene expression treated with 4 Gy radiation were 14 d for supercoiled plasmid and 21 and for linearized plasmid, while in control group, the time was 12 d. Middle-dose radiation combined with CD and 5-FC could kill 99 percent of colorectal carcinoma cells, while in the control group, 5-FC only killed 15 percent of colorectal carcinoma cells which were transduced with CD gene.</p><p><b>CONCLUSIONS</b>X-Ray combined with suicide gene therapy may be used as a promising method for treating colorectal neoplasm.</p>

In situ transduction of cytosine deaminase gene followed by systemic use of 5-fluorocytosine inhibits tumor growth and metastasis in orthotopic prostate cancer mouse models / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the antitumor and anti-metastatic effect of in situ transduction of adenovirus encoding cytosine deaminase (AdCD) followed by the systemic use of 5-fluorocytosine (5-FC) in the orthotopic (o.t.) prostate cancer mouse model.</p><p><b>METHODS</b>The o.t. prostate cancer model of C57BL/6 mouse was developed by o.t. inoculation of RM-1 cells to the subcapsular area of the prostate gland. In situ transduction of the CD gene, followed by systemic use of 5-FC at a daily dosage of 300 mg/kg for 14 days, was performed two days later.</p><p><b>RESULTS</b>Compared with mice treated with Adbeta-gal/5-FC, 5-FC and PBS, mice of the o.t. model receiving in situ treatment of AdCD/5-FC had significant prolongation of survival and suppression of local tumor growth. More importantly, pathological observations showed that metastatic activity occurred in all mice of the PBS, 5-FC and Adbeta-gal groups including metastasis to the iliac lymph node (10/10, 10/10, 10/10) and the lung (8/10, 7/10, 7/10). However, only two out of ten had iliac lymphatic metastasis in the AdCD/5-FC group with no systemic or preaotic lymphatic metastasis, suggesting a strong metastatic inhibitory effect.</p><p><b>CONCLUSIONS</b>In situ transduction of AdCD followed by systemic use of 5-FC leads to the inhibitory effect on tumor growth and metastatic activity in the o.t. mouse model of prostate cancer. Clinically, it may be possible to treat metastatic or recurrent prostate cancer with a novel gene therapy using in situ injection techniques in future.</p>

Study on the in vivo killing activity of YCD/5-FC gene therapy system on K562B cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To elucidate the killing activity of yeast cytosine deaminase/5-fluorocytosine (YCD/5-FC) gene therapy system on gene-transferred tumorigenic cell line K562B in vivo.</p><p><b>METHOD</b>K562B cell was infected with high titer virus and a gene transferred cell clone, YCD-K562B, was selected. Twelve male SCID mice of 4 week old were divided into 2 groups at random and both YCD-K562B and K562B cells were implanted to each mice. 5-FC or saline was given i. p for 10 days after tumor developed, and relative tumor volume was measured every 3 days. At the end of experiment, animals were sacrificed and the specimens were processed for histopathological examination.</p><p><b>RESULTS</b>At the end of experiment (21 days after tumor cell implantation), the relative tumor volume of the 4 groups were: YCD-K562B + 5-FC 2.922 +/- 0.581, YCD-K562B + saline 24.434 +/- 4.790, K562B + 5-FC 22.701 +/- 2.350 and K562B + saline 24.460 +/- 1.670; t-test analysis showed that 5-FC could kill cells (YCD-K562B) in vivo (P = 0.0001), but had no effect on the growth of gene-untransferred cells (K562B) (P = 0.096). In YCD-K562B + 5-FC group, relative tumor volume reduced in 3 approximately 6 days after treatment (the minimum was 0.681). Necrosis around artery could be found in the tumor of YCD-K562B + 5-FC group.</p><p><b>CONCLUSION</b>YCD/5-FC suicide gene therapy system has a significant in vivo killing activity to gene-transferred tumorigenic YCD-K562B cell.</p>

Nucleoside deaminases activity levels in diabetes mellitus, possible new indices for early detection of diabetic nephropathy

The clinical value of assaying the activity of the nucleoside deaminases-cytidine deaminase [CD], guanosine deaminase [GD] and adenosine deaminase [AD] was studied in thirty-nine type I and fifty- three type II diabetic patients together with fifteen completely healthy subjects as controls. The changes were presented in both types of diabetes. The degree of control did not affect the AD activity levels. However, patients with complicated disease had lower activity levels of AD than uncomplicated diabetes. This suggested that AD deficiency in diabetes is at least in part attributing to decreased immunity in diabetic patients. The possibility of being associated with inheritance of type I and type II diabetes could not be excluded. The increased activity levels of CD and GD could arise from evident or latent nephropathy, where the activity levels of both enzymes were significantly high in patients with nephropathy compared with those without nephropathy. The correlation between these two enzymes with either duration of diabetes or fructosamine supported this hypothesis. Thus, these enzymes could be valuable new indices in the early detection of diabetic nephropathy

Adenosine deaminase activity in leprosy (a preliminary study).

Adenosine deaminase (ADA) activity was studied in 25 patients having different types of leprosy and 25 healthy volunteer as control. There was definite rise of ADA activity in BL (72.9 +/- 6.85), LL (56.7 +/- 3.35) and BT (39.1 +/- 8.28) which was statistically significant when compared to ADA activity in healthy control (9.7 +/- 0.53).