Expression And Significance Of P33 And PINCH In Brain Glioma

Objective: Gliomas are the most common intracranialtumors in the incidence of neurosurgery tumors. The currenttreatments had little effect on the behavior of these tumors.Therefore, firstly the molecular mechanism should be beated outalthough development of novel therapies is of great importance.As known, gliomas result from specific genetic mutations ofmany oncogenes and/or tumor suppressor genes. But theinactivations and alterations of them are not completelyclarified .These complex correlations of oncogenes and tumorsuppressor genes are awaited to be illuminated. Tumorsuppressor genes play an essential role in the mechanism ofresisting tumorigenesis. A novel tumor suppressor gene,P33ING1(ING, inhibitor of growth 1), although recently berecognized, cooperates with P53 dependently in the structureand function, facilitates cell apoptosis and restricts cell growth.But little studies have been undertaken on the expression ofP33ING1 in brain glioma tissues in vivo. Furthermore, gliomasparticularly GSM have the biological abilities of invasivegrowth, proliferation prosperity, high postoperative recurrenceand so on. The invasive processes involve tumor cells adhesion,proteolysis of ECM (extracelluar matrix) and cells migration.The root causes of invasion and metastasis are not elucidatedabsolutely. PINCH is a new adapter protein that functions atinteracting with the special protein-complexes and signaltransduction pathway as key convergence point. It maybeactivates the malignant proliferation and invasive growth oftumor, not to be investigated in glioma. In this report, P33ING1and PINCH were detected in order to further investigate theirbiological characters and the mechanism of oncogenesis andprogression in glioma from the transcription and protein level.As a result, these may provide some possible means and newthinking-ways for clinical therapy and novel drug discovery.Methods: 50 fresh samples of glioma which are excised atthe Second Affiliated Hospital, Hebei Medical University from2003 to 2004 were collected, and which included 28 cases ofMale and 22 cases of Female. The range of age was from 6years old to 76 years old (mean±SD, 45.2±11.7), and include 36cases of <60 years old, 14 cases of >60 years old.The range ofdisease course was from 5 days to 2 years (2 month).Preoperative chemiotherapy, radiotherapy andimmunosuppressive therapy were not performed to all of thepatients. The tumors were graded and classified according tothe WHO (2000) scheme and consisted of 10 cases of WHOgrade Ⅰ[ pilocytic astrocytoma (8), choroid plexus papilloma(2) ], 16 cases of WHO grade Ⅱ[ fibrillary astrocytoma (8),protoplasmic astrocytoma (3), oligodendroglioma (3), andCemistocytic astrocytoma (2) ], 18 cases of WHO grade Ⅲ[ anaplastic astrocytoma (16), and Anplastic ependymoma (2) ],and 6 cases of WHO grade IV [ glioblastoma multiforme (5),ependymoblastoma (1) ]. 8 cases of normal brain tissue used ascontrol groups were obtained from the patients with braintrauma who underwent inter-decompression. Each specimensplit into two pieces: one for RT-PCR: snap-frozen in liquidhitrogen and transferred to a -80 ℃freezer; the other forimmunohistochemistry staining: routinely processed 4%formamint-fixed, serial sections of 5μm were cut from theblocks. Then representative sections were stained with H&E inorder to comfirm the histopathological diagnosis.RT-PCR technique: Total RNA was prepared by using Tizolreagent. RNA yield and purity were measured byspctrophotometric determination at 260nm and 280nm. And itwas visualized on formaldehyde denaturing agarose gel. Second,unique cDNA primers were designed for amplification ofP33ING1 and PINCH from tissues by semiquantitative RT-PCR.Primers: P33ING1, 5'-GCGGCGGATGCTGCACTGTGT-3'(sense) and 5'-CGCCGTCGTCGTGGTCGTGGT-3'(anti-sense);PINCH, 5'-GCGCCCGCTGAGAAGATCGTGAA-3'(sense)and 5'-TCCCCTTTCAGCTCCCGTGCATC-3'(anti-sense). Allthe parameter of every circulation temperature were modulated.RT-PCR products are 314bp and 482bp respectively. Tird,products of RT-PCR were electrophoresed in 1.5% agarose.Then, P33ING1 mRNA and PINCH mRNA were evaluated by aGel-analyzer software package respectively. The photonintensity as a ratio (RI) against that of corresponding β-actinmRNA was the expression parameter.Immunohistochemical stains (S-P): The processes wereperformed to examine the expression of P33^ING1 and PINCH.The cytoplasm of P33ING1 (partly nucleus or cell membrane) andPINCH positive cells showed buffy staining. Scoring thepercentage of positive cells in at least five high-power fields(×400) and counting 1000 tumor cells every field, took it asLabelling index (LI,%). Assessment of P33^ING1 and PINCHexpression was based on the mean percentage of positivetumour cells and assigned to one of five categories: -:nopositive tumor cell; +:the percentage of positive tumor cells<30%;++:percentage of positive tumor cells 30%～60%;+++:percentage of positive tumor cells >60%.The software package SPSS 11.5 was used for statisticalanalysis. RI and LI are reported as mean±standard deviation.The corresponding tests were used to analysis the data. P<0.05was considered statistically significant.Results: 1. Expression of P33^ING1 at transcript level inglioma: 45 of 50 (90%) glioma cases were positive, and 100%positive expression in normal brain. The mean P33^ING1 mRNAexpression level in normal tissue 0.84±0.22 was much higherthan that in glioma 0.56±0.21. Both the positive ratio and thequantity in normal tissue were remarkably higher than those ingrade III ,IV (P<0.05).The difference was statisticallysignificant in every grade and their expression quantitydecreased with increasing grade progress of gliomas (P<0.01).Its expression was not related to gender, age and location inglioma. The percentage of PINCH expression was 66% in alltumor specimens and 25% in normal tissue. On the contrary,The mean PINCH mRNA expression level in glioma 0.72±0.29was much higher than that in normal tissue 0.34±0.08. Both thepositive ratio and the quantity in grade III,IV were remarkablyhigher than those in normal tissue (P<0.05). The RI of PINCHmRNA in grade I,II was outstandingly higher than that in gradeIII,IV(P<0.01). The difference was statistically significant andtheir expression quantity increased with increasing gradeprogress of gliomas. The expression was not related to gender,age and location in glioma. 2. The percentage of P33ING1positive cells expression was 90% in glioma and 100% innormal tissue. The expression trend is the same as mRNA, theLI of mean P33ING1 expression level in normal tissue 80.01±2.22were much higher than that in glioma 53.04±22.75(P<0.05).The percentage of PINCH positive cells expressionwas 76% in glioma and 37.5% in normal tissue. As the mRNAexpression trend, the LI of mean PINCH expression level inglioma 49.56±21.86 were much higher than that in normal tissue20.36±8.20 (P<0.05). The difference was statistically significant.3. the correlativity:Both the RI and LI of P33ING1 expressionhave negative rank correlations with PINCH expression(-1