Expression And Action Of Keratinocyte Growth Factor And Its Receptor In Uterine Cervix Cancer

Objective: Keratinocyte growth factor(KGF), originally isolated from humanembryonic lung fibroblasts, is a member of the fibroblast growth factor family. Itis synthesized and secreted by stromal ceils and acts on epithelial cellsspecifically through a high-affinity receptor for KGF (KGFR). Therefore, KGF isconsidered to be a unidirectional paracrine effector that regulates normalepithelial cell proliferation, migration and differentiation. Among FGFs, KGF isunique because it interacts only through the KGF receptor (KGFR, also known asFGFR2Ⅲb), which is expressed exclusively by epithelial cells. Keratinocytegrowth factor receptor (KGFR), possesses intrinsic tyrosine kinase activity, is asplice variant of FGFR-2. KGFR is expressed in many types of epithelial cell andis activated with four known ligands [FGF-1, FGF-3, FGF-7 (also knownas KGF)and FGF-10]. Over the past year, studies have demonstrated that KGF,KGFR isassociated with epithelial tmour progression.It promotes cancer cell proliferation,migration and inhibition of apoptosis .Recent study has been reported that KGF,KGFR showed 8.92 and 12.83-fold higher expression levels in cervicalcarcinoma cells than in normal samples respectively. It is postulated that KGF,KGFR could involved in the invasive growth of malignant cervical squamousepithelia. It is now widely accepted that human papilloma viruses (HPVs) are the primary causal agents for the development of preneoplastic and malignant lesionsof the uterine cervix. The discrepancy between relatively high infection rates withpersistent high-risk HPVs and the relatively low rates of HPV-positive womenwho actually develop carcinoma of the cervix suggests that development ofcervical cancer is a multistep process, one that can not entirely be explained byinfection with certain high-risk HPV types. An important characteristic of tumormalignancy is the ability of proliferation, migration and inhibition of apoptosis oftumor cells. Several studies have demonstrated that KGF, KGFR effect onproliferation, migration, invasion and inhibition of apoptosis of tumor cell.However, information is limited on expression of KGF,KGFR in cervicalcarcinomas and KGF, KGFR functions have not been determined in uterinecervix cancer. The objective of this study is to investigate the expressions ofKGF, KGFR in normal cervical tissues, cervical cancer tissues, Hela cells, CaSkicells and action of keratinocyte growth factor on proliferation, migration andapoptosis of the Hela, CaSki cells.Methods:1,Immunohistochemical staining was performed to examine the localization ofKGF,KGFR in 22 normal cervical tissues and 31cervical cancer tissues.2,Reverse transcriptase polymerase chainreaction (RT-PCR) method was used todetermine gene expressions of KGF and KGFR in Hela, CaSki cell; ELISA andWestern blot method was used to determine the protein expressions of the KGFand KGFR in Hela, CaSki cell.3,~3H-Thymidine incorporation method was used to determine effect ofrecombinant human KGF on Hela, CaSki cell proliferation; the effects anti-KGFand anti-KGFR on on Hela, CaSki cell growth were measured by 3H-Thymidine incorporation and MTT.4,Millicell-PCF was used to determine effect of recombinant human KGF,anti-KGF and anti-KGFR on Hela, CaSki cell migration.5,Flow cytometry (FCM), ~3H-Thymidine incorporation method was used todetermine effect of recombinant human KGF on apoptosis of Hela, CaSki cell.Results1,Immunohistochemical analysis in normal cervical tissues: KGF, KGFR wasnot expressed in epithelial cells. It was regard as negative In normal cervicalepithelial cells. KGF was detected in cervical carcinoma cells in 14 of 31(45％);KGFR was detected in cervical carcinoma cells in 22 of 31 (71％). Expression ofKGF in the cervical cancer cells is positive correlation with expression KGFR;The positive expression rate of KGF, KGFR relate to depth of tumour invasionand clinical stage. The positiveexpression rate of KGF relate to tumor-celldifferentiation .There was no relationship between the expression of KGFR andtumor-cell differentiation. KGF, KGFR was also detected in normal cervicalstromal tissue. The same result detected in cervical cancer stromaltissues. however, expression of KGF, KGFR in two stromal tissues was notdifferent(P＞0.05).2,Expression KGF, KGFR in Hela, CaSki cell: both KGF/KGFR gene andprotein were expressed in Hela, CaSki cell; We could assume the formation ofautocrine loops of KGF and KGFR within the Hela, CaSki cell.3,Effection of KGF, KGFR on Hela, CaSki cell proliferation: Hela, CaSki cellwas stimulated to proliferate in response to recombinant human KGF; Treatmentwith Recombinant human KGF resulted in a dose-dependent increase in theproliferation of Hela, CaSki cells. The proliferation of Hela, CaSki Cell was inhibited by KGF antibodies and KGFR antibodies(P＜0.05).4,Effection of KGF, KGFR on Hela, CaSki cell migration: Treatment with100μl (50ng/ml) recombinant human KGF, cells number under Millicellpolycarbonate microporous membrane were increased than that of control one(P＜0.05); However treatment with anti-KGF, anti-KGFR resulted in decrease incells number. The cells under polycarbonate microporous membrane weredecrease than that of control one (P＜0.05). The action of KGF on CaSki cell ismore than that of Hela cell.5,Effection of KGF on Hela cell apoptosis: The apoptotic rates induced by DDPor KGF+DDP were 20％and 10％, respectively. So the apoptotic rates induced byDDP could be inhibited by KGF; When the Hela cells were treated with 10μg/mlDDP or 100ng/ml KGF+10μg/ml DDP and the proliferation of Hela cell wasdetected by ~3H-Thymidine incorporation method. The proliferation of Hela cellwas inhibited more effectively in the absence of KGF: KGF inhibited theinduction of apoptosis by DDP in Hela cells.Conclusions:1,The expression of KGF and KGFR in the normal cervical epithelial cells isnegative and positive in cervical cancer epithelial cells. Expression of KGF in thecervical cancer cells is positive correlation with expression KGFR; The positiveexpression rate of KGF, KGFR relate to depth of tumour invasion and clinicalstage. The positive expression rate of KGF also relate to tumor-celldifferentiation. These results seem to indicate that KGF and KGFR may beinvolved in proliferation and invasion of cervical cancer.2,Hela and CaSki cell express KGF and KGFR. There is autocrine loops of KGFand KGFR within the Hela, CaSki cell. 3,Recombinant human KGF, autocrine KGF and KGFR, all of them effect onproliferation, migration,invasion and inhibition of apoptosis of Hela and CaSkicell.