Alteration Of Cell Division Mode And Numb Expression In The Carcinogenesis Of Cervical Squamous Cell Carcinomas

Background Uterine cervical cancer is the second most common gynecological malignancy among women worldwide. Although it is well known that the infection of HPV is the critical etiologic factor of cervical carcinomas, the mechanisms of the carcinogenesis procedure are still largely unknown.During the course of tumor progression, cancer cells acquire a number of characteristic alterations. These include the capacities to proliferate independently of exogenous growth-promoting or growth-inhibitory signals, to invade surrounding tissues and metastasize to distant sites, to elicit an angiogenic response, and to evade mechanisms that limit cell proliferation. Above all, cancer cells gain the ability to proliferate unlimitedly. It is no doubt that cell proliferation should achieve through cell mitosis. Since recent decades, studies in invertebrates and vertebrates embryonic neurogenesis have revealed that there are two types of cell division modes— symmetric division and asymmetric division. They found that symmetric division wasproliferative, the two daughter cells always divided again after mitosis. However, asymmetric division was differentiative, the one daughter cell always reentered the cell cycle but the other began to differentiate. So in brief, symmetric division can make cell gain the ability to proliferate repeatedly, and it is still a puzzle whether such unlimited proliferating ability of cells during tumor progression is acquired through an increase of symmetric divisions? And whether the alteration of regulatory molecules of cell division modes plays a critical role in carcinogenesis?Researches about the mechanisms regulating cell division modes have shown that different cell cleavage orientation produces different cell division mode. Horizontal divisions —with the angles between cell cleavage plane and the apical surface ranging from 60° to 90° —are symmetric divisions, while vertical divisions with the angles ranging from 0° to 30° are asymmetric divisions. Such results provide us a simple morphological method for determining these two types of cell division modes. However, further studies have discovered that the substance of different cell division mode is because the distinct distribution of cell fate determinants during mitosis. Numb is one of the most important cell fate determinants. Equal separation of Numb to two daughter cells in mitosis results in symmetric division. Otherwise, if Numb is distributed to only one daughter cell, asymmetric division occurs. Numb is evolutionally conserved among various species, and there are four human Numb isoforms because of differential mRNA splicing. Studies have shown us that Numb-PTBL including h-Numbl and h-Numb2 localizes to cell membrane, and Numb-PTBS including h~Numb3 and h-Numb4 diffuses in cytosol and nucleus. Distinct human Numb-PRR isoforms play different role inregulating differentiation versus proliferation. Numb-PRRL including h-Numbl and h,-Numb3 promotes cell proliferation, whereas Numb-PRRS including h-Numb2 and h-Numb4 enhances differentiation.On the foundation of the similarity between cancer cell division mode and symmetric division, we used cervical squamous cell carcinomas (CSCC) as our research objects, and then by measuring cell cleavage angles and detecting Numb expression we investigated if the alteration of cell division mode participated in the carcinogenesis of CSCC and its possible molecule mechanisms.Materials and Methods We used 178 patients' paraffin embedded cervical tissue sections—including 50 cases of ISCC, 21 cases of CINI, 59 cases of CINII—III and 48 cases of normal cervical controls—to determine the cleavage angles of basal cells in mitoses of cervical squamous epithelia through light microscope (except ISCC cases for being unable to check the cleavage angles) and to detect Numb expression using immunohistochemical staining. Then we used RT-PCR to detect distinct Numb-PTB and Numb-PRR mRNA splicing isoforms respectively in another 65 patient' s fresh cervical tissues including 37 cases of ISCC and 28 cases of normal cervical controls. Semiquantitative analysis of Numb-PRR isoforms mRNA was also done with beta actin as the internal standard. At last, further detection of Numb-PRRL mRNA utilizing in situ hybridization in paraffin embedded cervical sections of these aforementioned 65 cases was undertook in this study.Results Statistical analyses showed that the distribution of the cleavage angles differed significantly between CINII-III cases and CINI cases (P= 0.004) as well as between CINII-III cases and normal cervical controls (P= 0.013). No significant difference was found between CINIcases and normal cervical controls(P=0.195). However, the data exhibited that there was an increase of basal cells with horizontal divisions but a decrease of vertical divisions in CINI cases. As to CINII-III cases, the proportion of horizontal divisions reduced while oblique divisions and vertical divisions rose again. Immunohistochemical expression of Numb was found in the ectocervical epithelia in all of the ISCC, CINII-III, CINI and normal cervical controls. There was cytoplasic and membrane staining in all squamous epithelia as well as a slight nucleus staining of some areas. Even for cells during mitotic phases, no cells with asymmetric localization of Numb were found. But the expressing intensity of Numb had a tendency to be stronger gradually from normal cervical controls to ISCC cases (P<0. 001), expect that there was no significant difference between the CINI cases and normal cervical controls (P=0. 828). Results of RT-PCR showed that both Numb-PTB~L and Numb-PTB~S mRNA were detected in all these 65 cases. Numb-PRR~L and Numb-PRR~S mRNA could also be amplified in all these cases except Numb-PRR~L mRNA was absence in one normal cervical control. Semi quantitative analysis of Numb-PRR isoforms mRNA exhibited that the band intensity of Numb-PRR~L mRNA was 1.026 + 0.683 and 0.515 ±0.354 for ISCC cases and normal cervical controls respectively when normalized by beta-actin expression level. There was a significantly higher expression of Numb-PRRL mRNA in ISCC cases than in normal cervical controls (P<0. 001). However, the Numb-PRRS mRNA expression between the two groups has no significant difference (0. 888 ±0.610 versus 0.854+0.515, /M). 812). Utilizing in situ hybridization in these 65 cases, positive cells expressing Numb-PRR1 mRNA were found in cervical squamous epithelia sporadically or diffusely but not in stroma, and they appeared to be red staining in cytosol around nucleus.There was a significantly higher expression of Numb-PRRL mRNA in ISCC cases than in normal cervical controls (/M). 015).Conclusions All these results suggest us: 1. In the initial stage of the carcinogenesis of CSCC, cells with carcinoraatous changes may acquire an increase of symmetric divisions by altering cell cleavage orientation and then these cells proliferate. 2. Human cervical squamous epithelia express Numb protein. An increase expression of Numb-PRRL isoforms and the following up-regulation in ratio between Numb-PRRL and Numb~PRRs may be one of the mechanisms of the genesis of CSCC.