Long-term stable expression of antisense cDNA of cyclin B1 profoundly inhibits the proliferation of tumor cells and suppresses tumorigenicity in implanted mice

Background Cyclin B1 （CLB1） is necessary for mitotic initiation in mammalian cells and plays important roles in cancer development. Therefore, a potential strategy in cancer therapy is to suppress the activity of CLB1 by delivering antisense constructs of CLB1 into tumor cells. In previous CLB1 studies, antisense constructs with a short half life were often used and these constructs might not persistently inhibit CLB1. Methods We successfully created a recombinant plasmid encoding the full-length antisense cDNA of mouse cyclin B1 （AS-mCLB1） and transfected this construct to the murine Lewis lung carcinoma （LL/2） and CT-26 colon carcinoma （CT-26） cells. We isolated clones of LL/2 and CT-26 transfectants with stable expression of AS-mCLB1. Reverse transcriptional polymerase chain reaction （RT-PCR） and Western blot were applied to detect the expression of the mRNA and protein levels of CLB1. To further test the efficacy of this strategy in vivo, AS-mCLBl-expressing LL/2 and CT-26 transfectants were implanted into mice. Results We found the expression of the mRNA and protein levels of CLB1 decrease in these transfectants. The inhibition of CLB1 caused prominent G1 arrest, abnormal morphology, retarded cell growth and an increase in apoptosis. In AS-mCLBl-expressing LL/2 and CT-26 transfectants implanted mice, tumorigenicity was effectively suppressed compared with the controls. In addition, the expression of AS-mCLB1 also significantly increases the survival duration of implanted animals. Conclusion AS-mCLB1 is likely to be useful in future cancer therapy, which may be associated with its ability to down-regulate the expression of CLB1 and then induce G1 arrest and apoptosis in tumor cells.

Effect of the Expression of HSP 70 Gene on the Fertility of Sorghum 1

It was found that the Cytoplasmic Male Sterility (CMS) line of sorghum 3197A (3A) can be induced to become fertile by heat shock treatment, whereas the maintainer line 3197B (3B) is fertile, whether it is heat shocked or not. During the pollen mother cell (PMC) stage, with heat shock treatment, the 3A line changed into fertile from sterile, and five protein bands: 70kD (HSP 70 ), 31kD, 24kD, 18kD and 16kD were found. And identical bands were also appeared in 3B line with similar treatment. By comparing the amount of protein in mitochondria during ear stage of 3A sorghum, the amount of protein increased by 2.7 times, and was almost as much as that in 3B. A comparison of the heat shock proteins (HSPs) of mitochondria in 3A and 3B lines revealed that HSPs were encoded by the nuclear DNA and transported to the mitochondria. Moreover, HSPs were more heat stable in 3B than in 3A line, and this may be related to the stability of fertility in 3B line. To demonstrate the correlation between fertility and HSPs, DNA complementary to HSP 70 was injected into ears of 3A and 3B line. It is found that under normal conditions, there was no HSP 70 mRNA in 3A line, whereas after heat shock this mRNA appeared, and the antisense cDNA could combine with the target RNA of 3B either dealt with normal temperature or heat shocked. These results are discussed within the frame of fertility versus sterility in sorghum.

Androgen-independent growth in LNCaP cell lines and steroid uridine diphosphate-glucuronosyltransferase expression

Aim: To investigate the mechanism of androgen-independent growth of prostate cancer after androgen ablation in LNCaP cells and the effect of glucuronidation activity. Methods: To establish androgen-independent growth in prostate cancer LNCaP-SF, continuous passage was performed in androgen-stripped medium and the cells were evaluated for glucuronidation activity. The expression vector of antisense uridine diphosphate glucuronosyltransferase (UGT) 2B15 cDNA was also constructed and evaluated. Results: LNCaP-SF lead to a higher expression in UGT2B15 and their glucuronidation activity is 2.5 times higher than that of LNCaP cells. Significantly fewer LNCaP and LNCaP-SF than control were transfected with the antisense UGT2B15 cDNA, suggesting that UGT2B15 plays an important part in the glucuronidation activity of androgens in both cells. Conclusion: The alteration of UGT2B15 expression in LNCaP-SF cells is proposed as a biological characteristic involved in the growth of hormonerefractory prostate cancer.

Resistance of SKW6 cell to apoptosis induction with anti-Fas antibody upon transduction of a reverse fragment to a cDNA encoding human 6A8 a-mannosidase

The effect of transduction with a reverse fragment to a cDNA encoding human 6A8 α-mannosidase on apoptosis induction of human B cell line SKW6 by anti-Fas antibody was tested. Apoptosis-inducer of anti-Fas monoclonal antibody was used to induce apoptosis in SKW6 cells. Giemsa's staining, Annexin-V-FLUOS staining and DNA ladder test were used to determine the events of apoptosis. Indirect immunofluorescent staining with anti-Fas antibody was performed to detect the surface Fas expression. In a time-course test of 12, 24 and 36 h for apoptosis induction by anti-Fas antibody, DNA ladder was observed in the wild-type SKW6 cells in a time-dependent fashion. Mock transduction had no effect on DNA ladder production. However, no DNA ladder was detected in the rAAV-antisense 6A8 cDNA-transduced SKW6. Results from Annexin-V-FLUOS staining on anti-Fas antibody-treated cells revealed that the staining-positive rate in the rAAV-antisense 6A8 cDNA-transduced SKW6 cells was decreased in comparison to that in the wild-type and the mock-transduced cells. Giemsa's staining observation showed that the number of dying (with apoptotic bodies) and dead cells was reduced in the rAAV-antisense 6A8 cDNA-transduced SKW6 cells in comparison with that in the wild-type and the mock-transduced cells upon anti-Fas antibody induction. The transduction did not affect the expression of Fas molecular on cell surface. 100% cells in all the groups showed Fas expression. The SKW6 cells became resistant to apoptosis induction by anti-Fas antibody upon transduction with a reverse fragment to a cDNA encoding human 6A8 α-mannosidase. The transduction did not affect the expression of Fas molecule on cells.