[Construction of pMSCV recombinant retroviral vector containing polo-like kinase 3(plk3) cDNA and its effects on cell proliferation].
OBJECTIVE
To construct pMSCV retroviral vector (RV) containing Polo-like kinase 3(plk3) cDNA, a new member of Ser/Thr protein kinase family, and to study the function of plk3 gene by introducing it into human K562 and HL60 cells.


METHODS
plk3 cDNA was sub-cloned into retroviral vector pMSCV-puroR to generate pMSCV-plk3-puroR, pMSCV-puroR being used as the control. RVs were generated by introducing RV vectors into 293T-Amphotropic packaging cells. Titers of RV were measured by NIH3T3 cells with Large-scale-real-time titration method (LaSRT). K562 and HL60 cells were infected with pMSCV-plk3-puroR or pMSCV-puroR viral supernatant by flow-through transduction method, respectively. Following 3-day selection with 3 mg/L puromycin, K562-plk3-puroR, HL60-plk3-puroR, K562-puroR and HL60-puroR cells were obtained. PCR was used to identify the integration of plk3 gene in K562-plk3-puroR and HL60-plk3-puroR cells. Transduction rates were determined by clone-forming ability against puromycin in semi-solid culture medium. Cell growth curve was measured by MTT colorimetry, the cell cycle and apoptotic analysis were detected by flow cytometry, etc.


RESULTS
Virus titers generated was (1.31+/-0.65)x10(9)TU/L. The K562 and HL60 transfection efficiency reached 82.3%+/-3.70% and 62.9%+/-4.94%, respectively (n=3). After 3 d puromycin selection, purified K562-plk3-puroR and HL60-plk3-puroR cell were obtained. The proliferation of K562 and HL60 cells was slowed down by plk3 gene transduction. Compared with wild type K562 cells: (1) when cultured in complete DMEM medium, more K562-plk3-puroR cells were observed in G(0)-G(1), 49.7%+/-3.38% vs 43.9%+/-2.34% (P=0.03); (2) when cultured in serum-free DMEM medium for 10 h, fewer K562-plk3-puroR cells were observed in S phase, 43.6%+/-3.74% vs 54.5%+/-1.52% (P=0.02) with lower apoptosis rate, 3.4%+/-0.37% vs 1.3%+/-0.31% (P=0.01).


CONCLUSIONS
Introduction of plk3 gene into tumor cells with RV vectors can slow down cell proliferation, prevent cells into cell cycle and protect cells from apoptosis in serum-free culture.
Jiang
Shan
S
Department of Pathology, Nanjing Medical University, Nanjing 210029, China.
Feng
Zhen-qing
ZQ
Jiang
Qian-li
QL
Li
Yu-hua
YH
Peng
Tao
T
Yin
Hong
H
D000818:Animals; D002453:Cell Cycle / Q000235:genetics; D049109:Cell Proliferation; D018076:DNA, Complementary / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D018922:HL-60 Cells; D006801:Humans; D020014:K562 Cells; D051379:Mice; D041681:NIH 3T3 Cells; D016133:Polymerase Chain Reaction; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000502:physiology*; D025521:Tumor Suppressor Proteins; D000097868:Polo-like Kinases
D018076:DNA, Complementary; D017346:Protein Serine-Threonine Kinases; D025521:Tumor Suppressor Proteins; C486256:PLK3 protein, human; D000097868:Polo-like Kinases