Carcinoma of the uterine cervix emerges from a defined series of prenoplastic lesions with increasing cellular dysplasia referred to as cervical intraepithelial neoplasia (CIN) grade I, II and III 1. A large body of knowledge supports the view that high risk HPV types (HR-HPV) are strongly associated with invasive squamous cell carcinoma (ISCC) and its precursors as they have the ability to transform normal cervical cells into neoplastic ones 2. HPV 16 and 18 are the most commonly detected HR-HPV types in these lesions, and therefore their detection was proposed as a useful surrogate marker to diagnose cervical dysplasia and carcinoma in situ (CIS) 23.

Progress in cervical cancer research provided evidence that integration of HR-HPV DNA into the host cell genome results in elevated expression levels of the E6 and E7 proteins with subsequent interaction between these oncogenic proteins and the cell cycle machinery 24. Expression of E6 and E7 proteins of HR-HPV types induces immortalization of cells through their inhibitory effects on the tumor suppressor proteins pRb and p53; respectively, altering the cell cycle control and leading to chromosomal instability 2. Undermining of pRb growth-inhibitory role with release of E2F transcription factors render the cells independent of mitogenic stimuli 5, whereas inactivation of the p53 precludes its role as one of the major factors controlling cell proliferation since it arrests the cell cycle in response to DNA damage or direct the damaged cell to an apoptotic pathway. This function is abolished via p53 degradation by the E6 protein of oncogenic HPV types or via cytoplasmic sequestration by complexing to mdm-2 protein 6.

However the E6-p53 and E7-Rb model is not sufficient to inevitably produce cervical carcinoma although it has resulted in the identity of the viral gene's actions on numerous cellular proteins and processes normally involved in cellular growth and proliferation. This is evidenced by the spontaneous clearance of HPV infection and the long delay between the onset of persistent infection and the emergence of malignancy. Recent studies show an association between HR-HPV types and cell cycle regulators 27. The cell cycle is governed by a family of cyclins, cyclin dependent kinases (CDKs) and their inhibitors (CDKIs) through activating and inactivating phosphorylation events. Attention has been focused on altered expression of G₁ cyclins and Cdks because the major regulatory events leading to cell proliferation and differentiation occur within the G₁ phase of the cell cycle. The D-type cyclins reach maximum levels of expression and form functional kinase complexes with CDK4 or CDK6; during the mid-G₁ phase, whereas cyclin-E is expressed and associated with Cdk2 in an active complex near the G₁-S boundary 58. Active CDK/cyclin complex can be regulated by binding to CDKI (p16^INK4A, p21^waf1 and p27^KIP1) and inhibit cell cycle progression from G1 to S phase 7.

Deregulation of p16^INK4A, p21^waf1 and p27^KIP1 has recently been reported in various human tumors. In HPV-associated cervical carcinoma, the expression and function of these proteins is supposed to be impaired by the action of viral oncopoteins E6 and E7 9. However, it is unclear how and when factors that are innate to the HPV-infected cells including genetic aberrations launch the host cell into an irreversible progression to cancer 7.

Therefore, we attempted to assess the contribution of cyclin D1, cyclin E, CDK4, p53, mdm-2, p21^waf, Rb, p27^KIP1, p16^INK4A and Ki-67 expression to the development of HPV16/18-associated cervical carcinoma and to determine at what stage of carcinogenesis these aberrations start to manifest. The prognostic value of these aberrations was also investigated in relation to the standard clinicopathological prognostic factors and overall survival.

The present study is the first to investigate the role of a large panel of cell cycle regulatory genes in Egyptian patients with cervical carcinoma both at the gene and protein levels. The studied population was homogeneous since all patients with CIN and ISCC were positive for HPV16 and/or 18. Tissue samples representing the different stages of transformation of normal cervical epithelium into ISCC have been analyzed for the possible role of the studied markers in etiopathogenesis of cervical carcinoma and their prognostic value.

It has been shown that the E6 and E7 proteins of HR-HPV types disrupt cell cycle checkpoints, particularly affecting CDKI linked to the G1- and G2-checkpoints with consequent accumulation of genetic aberrations 19. To our knowledge, the role of cyclins, CDKs and CDKI in ISCC and its precursor lesions is not well defined yet. Moreover no single study has investigated the contribution of these proteins together and in association with other cell cycle related genes such as p53, Rb and mdm-2. In the present study aberrant expression of CDKs and their inhibitors were detected at several points during the transformation of HPV-infected cervical epithelium.

Cyclin D1 amplification and protein overexpression were found in 46.5% and 41.9% of our ISCC cases as well as in18.4% and 13.15% of CINIII cases only. Our results are in agreement with Nicholas et al. 19 and Cheung et al. 15. In contrast, Bae et al. 20 reported reduced cyclin D1 mRNA and protein expression in cases of CIN and ISCC compared to NCT. It is difficult to explain the controversy between our results and those of Bae et al. 20. However, their study is one of the very few studies showing reduced cyclin D1 in cervical carcinoma. Moreover, they reported increased cyclin D1 expression with increasing severity of the lesion from high grade squamous intraepithelial neoplasia (HSIL) to ISCC which makes a higher expression in normal epithelium compared to the neoplastic one unlikely.

Our study shows a stepwise increase in the expression level of CDK4 from normal to tumor tissues indicating an important role for CDK4 at an early stage of transformation of HPV- infected cervical epithelium. Our results confirm the few available reports in this context 158. Although it is well known that cyclinD1 is necessary for the activation of CDK, the concordance reported in the present study between CDK4 and cyclin D1 expression was 49% only. We also detected increased CDK4 expression in cases of CINI and CINII although these cases did not show simultaneous increase in cyclin D1. This could be explained by the involvement of other D-type cyclins such as cyclin D2 and 3 in the activation of CDK4 21. Alternatively, the D type cyclins may not be required at all for G1 progression in HPV-transformed cervical epithelium since binding of HPV E7 protein to Rb leads to release of E2F transcription factor omitting the role of cyclin D1 in cell cycle progression 19.

Our results denote an important role for cyclin E in the early stages of HPV-associated cervical carcinogenesis since protein overexpression and gene amplification were detected during progression from NCT into ISCC. We also noticed a significant association between increased cyclin E expression and reduced p27^kip1. Our data confirm the results of Dellas et al 21 who demonstrated a feed back inhibitory loop between cyclin E and p27^kip1. This suggests the presence of a synergistic effect between both genes which will eventually lead to enhanced progression through the cell cycle as a consequence of the proliferative effect induced by increased cyclin E expression and the loss of the inhibitory function of p27^kip1.

Inactivation of CDKIs (p27^kip1, p21^waf, p16^INK4A) via reduced expression was reported in various human tumors 22. In HPV-associated cervical carcinoma, the situation is less clear since some studies showed that the tumor suppressor activity of these proteins is overcome through the action of the viral oncogenes E6/E7 without any change in their expression level 22, others showed that this applies to p21^waf and p27^kip1 only whereas p16^INK4A is usually down regulated 222324. Moreover, the HR-HPV types were shown to impair the function but not the expression of the p21^waf and p16^INK4A by rendering them insensitive to cyclin-CDK complex formation whereas p27^KIP1 is usually down-regulated 24.

In the present work, a stepwise decrease in p27^kip1 expression and a stepwise increase in p16^INK4A were found in cervical epithelium as it progressed from normal to a neoplastic one. Our results regarding p27^kip1 expression are in agreement with previously published data 8242526. In contrast, Shiozawa et al., 27 reported in their study a strong p27^kip1 expression in normal cervical epithelia which was markedly reduced to a negligible level in CIN samples. However, in ISCC cases they reported an increased expression of p27^kip1 and demonstrated that the p27 protein was bound to cdk2 and cyclin E. Consequently, the authors concluded that, p27^kip1 expression may be involved in the growth regulation of NCT however aberrant function of the p27^kip1 may occur in ISCC of the cervix. Although we can not find a proper explanation for this discrepancy in the results, we assume that the mechanism proposed by Shiozawa et al. 27 might represent an alternative pathway for p27^kip1 inactivation in ISCC of the uterine cervix through binding and sequestration by cdk2 and cyclin E which render it inactive just as the case with p53 and mdm2.

In our study, p16^INK4A overexpression was detected during the early stages of cervical carcinogenesis although no mutation was reported in any of the studied cases. We therefore assume that inactivation of p16^INK4A gene in HPV-associated ISCC is possibly achieved via mechanism(s) other than gene mutations. Among which is the binding to- and sequestration by other cellular and/or viral proteins. Our results are consistent with Volgareva et al. 22 and Tringler et al. 28 who reported p16^INK4A overexpression in a high percentage of dysplastic and neoplastic lesions of the cervix uteri. They also mentioned that p16^INK4A could be used as a surrogate marker for early diagnosis of cervical carcinoma. However, our results regarding p16^INK4A gene mutations in ISCC contrast with Tripathi et al. 17 who reported p16^INK4A gene mutation in 15% of their ISCC cases. A difference in sampling methods, clinical and virological features of studied cases or a racial difference could be mentioned as possible explanations for the controversial results between the two studies.

An interesting finding in this study is the significant increase of p21^waf which was recognized at CINIII and ISCC cases only. Although it is expected that increased expression of p21^waf should suppress the progression of cells in the cell cycle and consequently suppress cell growth, all the cases that revealed p21^waf overexpression had a high Ki-67 positivity index. Several explanations could be mentioned in this regard including: 1) the interaction between HPV E7 oncoprotein and p21^waf which abrogates its inhibitory effect on cyclin/CDK4 activities, 2) the occurrence of checkpoint adaptation after constant stimulation, 3) altered or inhibited binding to the CDK4/cyclin D1 complex, or 4) mutations in downstream targets of p21^waf 381028.

Our study is the first to assess RB gene HZD and PM in addition to altered protein expression in cervical carcinoma and its premalignant lesions. We detected altered Rb expression in the early stages of cervical carcinogenesis whereas PM and HZD were reported in CINIII and ISCC only. Therefore, we assume that altered protein expression in the early stages of neoplastic transformation could be attributed to mechanism(s) other than gene deletion or silencing by PM. Degradation by HPV-E7, sequestration by other cellular proteins or inactivation by yet unidentified mechanism(s) could be mentioned in this context. Our results regarding altered Rb expression are consistent with the few available reports in this context 3828.

Since all ISCC cases are HPV-16 or 18 positive, it is not expected to find accumulation of the p53 nor the p53-induced proteins in these cases. However, elevated levels of p53 and p21^waf proteins were seen in a relatively high fraction of cervical cancer together with mdm-2 in CINIII and ISCC cases. Our data are comparable to Tsuda et al. 29 and Huang et al. 30 who found p53 overexpression in 11.4% and 40.5% of HSIL compared to 56.6% and 30.7% of ISCC cases; respectively. Previous studies on ISCC showed that p53 overexpression is not usually associated with gene mutation but with sequestration by cellular or viral proteins of which mdm-2 is a highly possible candidate. This is in agreement with our results where p53 mutations were detected in 4 cases only whereas mdm-2 over-expression was present in 51.2% of ISCC, 13.7% CIN.

In gynecologic oncology, valid prognostic factors are necessary to define biologically similar subgroups for analysis of therapeutic efficiency. In the present study cyclin D1, p27, p53 and a high Ki-67 showed a prognostic relevance as increased expression of cyclin D1, p53 and Ki-67 as well as loss of p27 were highly correlated with the standard clinicopathologic prognostic factors for ISCC and they were significantly associated with reduced OS. Our data regarding the prognostic value of cyclin D1 and p53 are in agreement with Bae et al. 20 and Huang et al. 30, respectively who demonstrated that cyclin D1 and p53 overexpression are associated with decreased disease free survival (DFS) and OS rates. Similarly, our data regarding p27 are consistent with previous reports 1622 which considered p27^KIP1 an independent prognostic factor that significantly correlates with poor survival in ISCC.

We conclude that, infection of cervical mucosa by HPV16 and 18 leads to deregulation of the cell cycle via altering the expression level of certain genes. Whereas p27^KIP1, cyclin E, CDK4, p16^INK4A and Rb aberrations are early events; cyclin D1, mdm-2, p53 and p21^waf aberrations occur late in cervical carcinogenesis (Figure 8). Together, these alterations lead to acceleration of the cell cycle with increased proliferation rate, as indicated by a high Ki-67 PI, and acquisition of more genetic damage. Our results also provide evidence that the application of immunohistochemical tests for p16^INK4A, cyclin E, p27^KIP1 could help in early diagnosis of cervical carcinoma since alterations affec