Mechanism Of Recombinant Adenovirus-Mediated Mutations Of Human Hypoxia Inducible Factor 1α Regulating Cell Proliferation And Apoptosis

IntroductionMammalian cells can respond differentially to wide ranges of oxygen concentration through alterations in both metabolic states and growth rates.A growing body of evidence indicates that hypoxia alters cellular proliferation in two distinct ways by apoptosis and cell cycle arrest.In transformed and/or immortalized cells,apoptosis is the predominant response to hypoxia,occurring via activation of a p53 tumor suppressor gene product-dependent pathway.These alterations are believed to take place through the direct action of p53 in apoptotic pathways.However,accumulation and transcriptional activation of p53 protein are observed only under near-anoxic conditions,not under hypoxia.Recent studies have suggested hypoxia/HIF-1αinhibit cell growth and promote apoptosis.HIF-1αcan induce apoptosis via two mechanisms.First,it can increase the stability of the product of p53.In environmental stress or DNA damage,p53 induces apoptosis by regulating proteins such as Bax,or it can cause growth arrest,which is mediated by p21WAF1/CIP1.Second,overexpression of BNIP3 induces apoptosis by binding to and inhibiting the antiapoptotic proteins Bcl-2 and Bcl-xL at hypoxia. Hypoxia induced apoptosis mediated by BNIP3 may be HIF-1αdependent because cells lacking HIF-1 cannot produce large amounts of BNIP3 and a reduced cell death rate is seen.Previous studies have indicated that hypoxia-induced cell cycle arrest is accompanied by a decreased activity of certain cyclin-dependent kinase(CDK) complexes and hypophosphorylation of retinoblastoma(Rb) protein,leading to inhibition of cell cycle progression.However,little information has been available as to the role of HIF-1 in the regulation of cell cycle machinery.Recent researches elucidated the critical role of HIF-1 in the regulation of cell cycle progression under hypoxia,by showing that the induction of HIF-1 activation prevents G1/S transition through at least two distinct mechanisms.First,the expression of two cyclin-dependent kinase inhibitors(CKIs),p21WAF1/CIP1 and p27KIP1,is increased transcriptionally in a HIF-1-dependent manner.Sustained expression of these CKIs is observed in wild-type cells,but not in HIF-1a null cells. p21WAFI/CIP1 and p27KIP1 suppress cyclin/CDK2 activity,and thus can reduce the ratio of phosphorylated to dephosphorylated Rb protein,resulting in cell cycle arrest at the G1/S interface.Collectively,these data clearly indicate that CKIs function with HIF-1 to regulate the cell cycle in response to hypoxia.Secondarily, HIF-1 may regulate cyclin E,but not cyclin A protein levels;both of these bind CDK2 and modulate its kinase(cyclin E/CDK2 kinase) activity dependent upon cell cycle phase.However,Cell division requires the coordinated assembly of cyclins and cyclin-dependent kinases that promote cell cycle progression through S phase and mitosis.p21WAF1/CIP1 is a cyclin-dependent kinase inhibitor preventing abnormal or premature proliferation by blocking cyclin kinase activity.Expression of p21WAF1/CIP1 increases when cells are damaged.In addition to controlling cell-cycle progression,p21WAF1/CIP1 participates in DNA repair and apoptotic processes. HIF-1 is a transcription factor and a key regulator of the hypoxic response.Many of its target genes are particularly relevant to cell proliferation,cell survival, apoptosis,angiogenesis,glucose metabolism,and so on.HIF-1 consists ofαandβsubunits,Theβsubunit is constitutively expressed,whereas activity of theαsubunit is sensitive to decreased oxygen levels.Regulation of HIF-1αby hypoxia is mainly at the protein level.Under normoxic conditions,hydroxylation at two prolyl residues (Pro402 and Pro564 in human HIF-1α,mainly Pro564) in the oxygen-dependent degradation domain(ODDD) of HIF-1αmediates interactions with the yon Hippel-Lindau(VHL) E3 ubiquitin ligase complex that targets HIF-1αfor proteasomal destruction;hydroxylation at an asparaginyl residue in the transactivation domain(TAD) of HIF-1α(Asn803 in human HIF-1α) blocks interaction of HIF-1 with the transcriptional coactivator CBP/p300.At normoxia,HIF-1αis unstable and is degraded rapidly.Previous researches on the function of HIF-1αare usually under hypoxic condition,and hypoxia itself can induce apoptosis.Several strategies have been used successfully for experimental activation of HIF-1α.For instance,deletion of the central ODDD results in a stable and constitutively active HIF-1αmolecule.An alternative approach has fused the N-terminal DNA and dimerization domain of HIF-1αto the TAD of herpes simlex virus VP16.In addition,the use of small-molecule inhibitors of the HIF hydroxylases will stabilize HIF and activate the transcriptional response.But these approaches have many disadvantages and can not mimic the nature of HIF-1αthoroughly.In the present study,to gain a further insight of HIF-1αfunctions in the regulation of the cell proliferation and apoptosis under normoxic conditions and explore the relationship between HIF-1αand p21WAF1/CIP1,we used a recombinant adenoviral vector expressing the human HIF-1αgene with point mutations at residues Pro564 and AsnS03.Both the critical Pro of the ODDD and the critical Asn of the TAD in tandem were replaced by Ala,which provided HIF-1αprotein with nearly full activity at normoxia.ObjectTo investigate the molecular mechanism of recombinant adenovirus -mediated mutations of human hypoxia inducible factor 1α(Ad-HIF-1α-Ala564-Ala803) regulating cell proliferation and apoptosis.MethodsRecombinant adenovirus Ad-HIF-1α-Ala564-Ala803 and Ad- lacZ were amplified in HEK 293A cells and adenoviral titer was determined by End-Point Dilution Assay. The infection efficiency was observed with X-gal staining.The expression of mRNA level of HIF-1αand p21WAF1/CIP1 in LoVo cells after infected with recombinant adenovirus under normoxia condition at desirable time points was performed by real-time PCR.Western blot was employed to verify HIF-1αand p21WAF1/CIP1 protein expression.The effect of recombinant adenovirus on cell proliferation was assessed by MTT assay.Flow cytometer was used to analyze the distribution of cell cycle.Hoechst 33342 flourescein staining was performed to observe the ratio of apoptosis of LoVo cells.Results(1)High-titer adenovirus were produced after amplified.The titer of Ad-HIF-1α-Ala564-Ala803 and Ad-lacZ was 4.0×10⁹ pfu/ml and 6.3×10⁹ pfu/ml, respectively.Lo Vo cells could be effectively infected by recombinant adenovirus in vitro,the infection efficiency has the dose - effect relationship with multiplicyties of infection(MOI).When MOI was 60,the infection efficiency was more than 90%.(2) The expression level of HIF-1αmRNA increased,accompanied by an increase in that of p21WAF1/CIP1 mRNA in recombinant Ad-HIF-1α-Ala564-Ala803 group.The expression of HIF-1αprotein was sustained at high level and that of p21WAF1/CIP increased gradually in Ad-HIF-1α-Ala564-Ala803 group,while both of the protein expression of HIF-1αand p21WAF1/CIP were at lower level in Ad-lacZ group.(3) In terms of MTT,there was no statistical significance between Ad-HIF-1α-Ala564-Ala803 group and Ad- lacZ group.The ratio of cells in G₁ phase increased and meanwhile that in S phase decreased significantly in Ad-HIF-1α-Ala564-Ala803 group compared with Ad-lacZ group,.(4) The ratio of apoptosis in LoVo cells was higher in recombinant Ad-HIF-1α-Ala564-Ala803 group than that in control group.Conclusion(1)Recombinant adenoviral vector can transfect HIF-1α-Ala564-Ala803 gene into LoVo cells with high efficiency in a dose -effect manner.(2) HIF-1αplays an important role in cell cycle and apoptosis.It might up-regulate p21WAF1/CIP1 to induce cell cycle arrest at G₁ phase and promote apoptosis.