The productive stage of the viral life cycle occurs in the terminally differentiating suprabasal layers of the epithelium. In these cells, the virus switches to a rolling-circle mode of DNA replication and amplifies its genome to higher copy number, expresses late genes encoding capsid proteins, and produces viral progeny (Flores et al, 1999).

As a rule, in benign warts and pre-neoplastic lesions, the HPV genome is not integrated into the host cell genome (it is maintained in episomal form). However, in true neoplasia, it is wholly integrated into the host genome, although some authors have shown the coexistence of episomal and integrated forms in cervical cancer (Kristiansen et al, 1994).

The site at which the viral DNA is opened during this process of integration is fairly constant, and occurs within the E1/E2 open reading frame of the viral genome. The E2 protein can function as either an activator or repressor of viral gene transcription depending upon the location of the E2-binding sites within the promoter region of the viral genome (Bernard et al, 1989; Phelps et al, 1987). However, as the E2 region of viral DNA normally represses the transcription of the E6 and E7 early viral genes, this interruption causes E6 and E7 protein overexpression (Finzer et al, 2002). After all, while HPV integration means the end of the viral life cycle due to the functional inactivation of large parts of the viral genome, it leads to de-regulated expression of the viral oncogenes E6 and E7.

Many experimental studies have investigated genomic HPV integration sites, and although integrated HPV genomes have been observed to show preferences for relatively few loci, no general integration hot spot has been identified. A recent review of integration sites confirmed that HPV integration sites are randomly distributed over the whole genome with a clear predilection for ‘fragile’ sites. No evidence supporting the targeted disruption or functional alteration of critical cellular genes by the integrated viral sequences has been unearthed.

III. Molecular pathophysiology in cervical cancer

In normal epithelium, basal cells are sequestrated from the cell cycle following migration into the suprabasal cell layers and are committed to terminal differentiation. During HPV infection, E7 and E6 are expressed in these cells, and abolish cell cycle progression restraints and delay normal terminal differentiation (Sherman et al, 1997). The effects of E6 and E7 on p53 and pRB and on many other cellular proteins have been extensively investigated, and significant alterations in cell cycle regulation can be attributed to the biochemical interactions between these two viral oncogenes and their respective cellular binding partners (Munger et al, 2001). Moreover, recently it was demonstrated that the E6 and E7 cooperatively disturb the mechanisms of chromosome duplication and segregation during mitosis, and thereby induce severe chromosomal instability (Duensing et al, 2001).

A. HPV E7 and retinoblastoma protein (pRb)

The critical role of HPV E7 protein in the malignant transformation of cervical epithelial cells is attributed to its effects on pRb, a member of the ‘pocket protein’ family, which also includes p107 and p130 (Vogelstein et al, 1993). The proliferation of normal human cells follows an orderly progression through the cell cycle under the influence of cyclin/cyclin dependent kinase (cdk) complexes. Each cyclin/cdk complex control a specific cell cycle transition, the key downstream targets of the G1 phase cyclin/cdk complexes are members of the pRb family, i.e., Rb, p107, and p103. During G1 progression, Rb is sequentially phosphorylated by cyclin D1/cdk4 and 6 and cyclin E/cdk2 complexes, whereas hypo-phosphorylated pRB represents the active form and inhibits S phase entry, thus the sequential phosphorylation of Rb inhibits the repressor activity of pRb. The repressor activities of pRB and of the related pocket proteins p107 and p130, are mediated by members of the E2F family of transcription factors. In G0/G1 hypophosphorylated pRB is bound to E2F. Since pRB encodes a transcriptional repressor domain, pRB/E2F complexes function as transcriptional repressors. Following phosphorylation by cdk in G1, pRB/E2F complexes dissociate and E2F acts as a transcriptional activator, which results in S phase entry (Weinberg, 1995) (Figure 2). However, the virally encoded protein, E7, binds to hypophosphorylated pRb and displaces the E2F transcription factor from pRb. Thus, binding to Rb by E7 is essentially for E7-induced cells transformation, because E7 proteins, which are associated with HPV strains with a low cervical cancer risk, show little or no affinity for pRb23.

As for the E7/pRB interaction, E7 targets pRb for ubiquitin-mediated degradation by the proteasome (Boyer et al, 1996). Moreover, it was suggested that the cellular transformation activity of E7 tightly correlates with its ability to degrade pRB (Jones and Münger, 1997). It appears that pRB degradation, not solely binding, is important for the E7-induced inactivation of pRb (Gonzalez et al, 2001).

In addition to regulation by phosphorylation and dephosphorylation, cdks are regulated by a group of functionally related proteins called cdk inhibitors. In differentiating epithelial cells, high levels of cdk inhibitors (p21^cip1 and p27^kip1) can lead to the formation of inactive complexes consists of E7, cyclinE/cdk2 and either p21 or p27. As a result, it appears that during HPV infection, the ability of E7 to stimulate S-phase entry is limited to a subset of cells with low levels of p21/p27, or to cells which express high enough levels of E7 to subvert the block to S-phase entry. However, recent studies suggest that E7 can also interfere with the activity of the cyclin-dependent kinase inhibitors p21^cip1 and p27^kip1 and thus override normal G1 checkpoint control (Jones et al, 1997; Zerfass-Thome et al, 1996). In addition, to cyclin/cdk complexes and cdk inhibitors, E7 can also associate with other proteins involved in cell proliferation, including histone deacetylases (Antinore et al, 1996) and components of the AP-1 transcription complex (Longworth et al, 2004), through p21 upregulation.

B. HPV E6 and p53

Although, E7 protein can independently immortalize various human cell types in tissue culture, efficiency is increased when E7 and E6 are coexpressed (Munger et al, 1989). Thus, E6 protein is believed to complement the role of E7 protein, and prevent apoptotic induction in response to unscheduled S-phase entry mediated by E7. However, the importance of HPV E6 in cancer appears to be primarily due to its effects on the cellular tumor suppressor gene, p53. The most commonly found alterations to p53 in cancers, such as, colon, breast, and lung cancer, are deletion, insertion, and point mutation (Bartek et al, 1990; Rodrigues et al, 1990; Takahashi et al, 1991). The p53 gene negatively regulates the cell cycle and “loss of function” mutation in p53 is required for tumor formation (Hollstein et al, 1991; Levine et al, 1991). Up to 99% of invasive cervical carcinomas have been found to contain HPV 16 or 18 DNA and in these few are found without evidence of HPV p53 mutations (Crook et al, 1992).

Normally p53 is transiently upregulated after DNA damage, which leads to cell cycle arrest in the G1 phase and apoptosis. This arrest allows time for DNA repair, and if repair is not possible, cells are committed to apoptotic death. p53 acts through downstream regulators, such as p21, which leads to cdk inhibition and the eventual blockade of Rb gene phosphorylation, thus preventing cell cycle progression. However, virally encoded E6 binds to a cellular ubiquitin/protein ligase, E6-AP, and simultaneously to p53, which results in the ubiquitination of p53 and its subsequent proteolytic degradation (Ferenczy et al, 2002) (Figure 3).

E6 proteins of high oncogenic risk HPVs, i.e., HPV 16 and 18, have a higher affinity for p53 than lower oncogenic risk types (HPV 6 and 11) (Crook et al, 1991; Lechner et al, 1994). Moreover, although the association between E6 and p53, and the inactivation of p53-mediated growth suppression and/or apoptosis have been well documented, this association can also lead to apoptosis via changes in the expressional levels of Bax and Bcl-2 family members (Selvakumaran et al, 1994). Consequently, the presence of E6 is considered to predispose the development of HPV-associated cancers, by allowing the accumulation of chance errors in host cell DNA to go unchecked. Moreover, the E6 protein of high-risk HPV types can also stimulate cell proliferation independently of E7 through its C-terminal PDZ-ligand domain (Thomas et al, 2002)

Both the p53 and Rb proteins interact with the double minute 2 gene (MDM2). P53 acts as a transcriptional activator of MDM2, whereas MDM2 acts in an auto-regulatory fashion by providing negative feedback to p53 transcription. MDM2 can also interact with Rb and restrain its action. One study found that MDM2 was overexpressed in up to 35% of cervical tumors, although this was not found to be correlated with HPV infection (Momand et al, 1992).

C. HPV E6 interaction with Bak

Apoptosis, or programmed cell death, triggers a series of events that lead to the expeditious elimination of unwanted cells. In activelyproliferating tissues, intrinsic apoptotic signaling is controlled by members of the Bcl-2 gene family, which are critical regulators of mitochondrial integrity and of mitochondria-initiated apoptosis. This family contains both anti-apoptotic (Bcl-2/Bcl-XL) and pro-apoptotic species (Bax, Bad and Bid). Thus, Bax promotes apoptosis whereas Bcl-2 represses apoptosis.

E6 has been shown to prevent apoptosis in both a p53-dependent and a p53-independent manner (Pan et al, 1995). p53 is one of the best known inducers of apoptosis, but its activity in HPV-infected cells is countered by viral E6 protein. Moreover, E6 can damage p53 by targeting it for ubiquitin-mediated proteolysis. However, E6 has been shown to prevent apoptosis in both a p53-independent and dependent manner. Recent research into HPV E6 oncogenic properties found that pro-apoptotic effect of Bak is a target of anogenital HPV E6 protein, and that this proceeds in a p53-independent manner (Thomas et al, 1998). E6 proteins from HPV-18, HPV-16 and HPV-11, can all bind Bak in vitro and stimulate its degradation in vivo, and this Bak downregulation was found to induce apoptosis via a E6AP-dependent process (Thomas et al, 1998). In fact, Bak was found to bind E6-associated protein (E6AP) in the absence of E6 unlike p53 (zur Hausen, 2000).

D. HPV and telomerase

Normal DNA replication leads to the erosion of chromosomal telomere termini, which leads to chromosomal instability and finally to cellular senescence. Senescence arises mainly as a result of telomere shortening (Horikawa et al, 2003).

Telomerase, a ribonucleoprotein that prevents telomere erosion, is expressed in certain cell types that undergo repetitive cell division. Moreover, telomerase activity is present in immortalized and cancer cells but not in normal cells. These relations indicate that telomerase activation is critically required for immortalization and malignant transformation. The loss of telomerase activity in normal cells results in gradual decrease in telomere length with successive cell cycle rounds (Veldman et al, 2001).

Telomerase activity is regulated at the level of telomerase reverse transcriptase (hTERT), the catalytic telomerase subunit. The ectopic expression of hTERT in cancer cells was found to cause cellular immortalization (Blasco et al, 2003). Recently it was shown that cervical carcinoma cells expressing integrated copies of the HPV genome show high telomerase activity (Baege et al, 2002; Singh et al, 2004). Moreover, it was demonstrated that expression of telomerase correlated significantly with the histological severity of the cervical disease (Park et al, 2003).

Multiple factors have been shown to up-regulate expression hTERT, including c-myc and viral oncoproteins (Greenberg et al, 1999; Wu et al, 1999). High-risk E6 is believed to induce telomerase activity during progression to malignancy (Klingelhutz et al, 1996), but the underlying mechanism remains elusive. However, it has been postulated that E6 interacts with c-myc and the c-myc/E9 complex synergistically to promote E6-mediated hTERT induction (Oh et al, 2001). Direct stimulation of hTERT promoter and prevention of the inhibitory effects of p53 have been suggested as an alternative mechanism of telomerase maintenance in cervical cancer cells (Seo et al, 2004).

E. Other oncogenes

Although, the over-expressions of E6 and E7 appear pivotal in the development of cancer, their expressing is not enough either for the immortalization of cultured human cells, or for malignant conversion. Rather, other specific genetic abnormalities are important during cervical carcinogenesis and the aggressiveness of cervical tumors. In addition to p53 and Rb, the ras family genes (K-ras, H-ras and N-ras) and c-myc oncogene might also have a role in the pathogenesis of cervical cancer (Baker et al, 1998; Bourhis et al, 1990; Dokianakis et al, 1998; Garzetti et al, 1998; Riou et al, 1987). Each of these genes has been reported to be overexpressed in cervical cancer and several of them have been associated with a poor prognosis.

The H-ras, K-ras, and N-ras genes are localized to chromosomes 11, 12, and 1, respectively, in humans. The three genes have a common structure and all encode 21-kDa (p21) protein, 189 amino acids long, with GTPase activity, which participate in cellular signal transduction. The activation of ras oncogenes by point mutations has been suggested to play an important role in the multistep process of carcinogenesis. The most frequent ras alterations in human cancer are mutations in codons 12, 13 and 61, which abolish p21 GTPase activity, thus rendering p21 constitutively activated. The over-expressions of ras genes has been reported in several human cancers, including those of the breast, colon, head and neck, bladder, and lung, and these have been associated with disease development. Elevated ras p21 protein expression was also reported in cervical tumors as opposed to benign or premalignant lesions. In in vitro studies, it was demonstrated that E6 and E7 can co-operate with activated ras oncogene to transform epithelial cells (Storey et al, 1993; Phelps et al, 1988). However, research data in literature remain contradictory on the role of ras and HPV oncogenes and their probable co-operation in the pathogenesis of cervical neoplasm. In a recent study, any point mutation in codons 12 and 13 of K-ras gene was not identified in tissue from high-grade cervical dysplasia and invasive cervical carcinoma (Pochylski et al, 2003).

Downregulated c-Myc (a protooncogene) expression, accelerates cell proliferation and cell transformation, and occurs frequently in human tumors (Brenna et al, 2002). In normal cells, Myc is required for cell cycle entry, and its overexpression results in apoptosis after growth arrest has been induced by an external insult. On the other hand, in cancers its overexpression cause cell cycle re-entry and acts as an angiogenic switch. The gene encoding c-Myc protein is located on chromosome 8q24, the locus within which the HPV 16 sequence is integrated, which suggests that HPV integration into the fragile c-Myc region may be important element of HPV-induced oncogenesis. Moreover, amplification and/or overexpression of the c-myc gene were frequently observed in advanced-stage cervical cancers, and these were shown to be associated with tumor progression (Covington et al, 1987). Moreover, c-myc overexpression was found to be related to a higher risk of distant metastases, which suggests that the activation of this proto-oncogene may lead to metastatic ability.

Alteration of the cell cycle regulatory gene CDKN2A also seems to be involved in HPV-associated carcinogenesis. The CDKN2A locus on chromosome 9p21 codes two proteins with different functions, the cyclin-dependent kinase inhibitor (CDKI) p16^INK4a and p14^ARF. While p16^INK4a prevents S-phase entry by inhibiting CDK4/6-mediated phosphorylation of retinoblastoma (RB), p14^ARF is a key trigger of p53 stabilization in response to oncogenic signaling (Serrano, 1993; Zhang, 1999). Although alterations of p16^INK4A and p14^ARF have been reported in some tumor types (Sharpless, 1999) the role of p14^ARF/p16^INK4a alteration in cervical cancer is less understood. Contradictory results have been reported about the expression of p16^INK4ain cervical cancer. If, on the one hand, reduced expression of p16^INK4ain cervical cancer has been reported in some studies (Nakashima et al, 1999), on the other hand, other studies have shown the overexpression of p16 ^INK4a in cervical cancer (Murphy et al, 2003; Sano et al; 1998). As for p14^ARF, expression level is poorly investigated in cervical cancer. Because p14^ARF expression is positively regulated by the E2F transcription factor and negatively regulated by p53 at the transcription level, HPV E6/E7 oncoprotein can be postulated to upregulate p14^ARF expression. In a recent study, it has been shown that p14^ARF and p16^INK4A were overexpressed in HPV-positive cervical cancers as a consequence of HPV E6 and E7 expression (Kanao et al, 2004).

F. E6 and E7 oncoprotein and chromosomal instability

Growing evidence indicates that a significant proportion of solid tumors show unstable aneuploidy, alteration in the number of chromosomes. It is shown that aneuploidy in cervical dysplasia is associated with the presence of high-risk HPVs (Rihet et al, 1996; Kashyap et al, 1998). Furthermore, a number of chromosome aberrations have already been involved in pre-invasive high-risk HPV-associated cervical lesions (Steinbeck, 1997; Bulten et al, 1998). In particular, aneuploid cervical intraepithelial lesions have a significantly higher risk for carcinogenic progression, which strongly supports the concept of genomic instability as a hallmark for cervical carcinogenesis (Bibbo et al, 1989)

The precise mechanisms underlying aneuploidy remain unclear, but it is believed to stem from an imbalance in chromosomal segregation (Pihan et al, 1998), which results from the unusual amplification of centrosomes (Chial et al, 1999) and/or the dysfunction of centromeres/kinetochores. In addition, certain viral oncoproteins have been implicated in the induction of chromosome copy number changes. In cervical cancer, it has been shown that expression of HPV E7 alone can be sufficient to induce a moderate level of aneuploidy, whereas the E6 oncoprotein renders cells prone to structural chromosomal changes (White et al, 1994). When both oncoproteins were co-expressed, an elevated level of aneuploid cells was found, indicating that HPV-16 E7 induces centrosome-related mitotic disturbances that are potentiated by HPV-16 E6 (Duensing S et al, 2000). Riley et al also reported that both E6 and E7 increased centrosome copy number and created invasive cancer when it acts in combination (Riley et al, 2003). In a more recent study, Schaeffer et al demonstrated that expression of either E6 or E7 interferes independently with the centrosome cycle, resulting in centrosome aberrations (Schaeffer et al, 2004).

IV. Genetic components in cervical cancer

A. Allelic variation

Certain HLA alleles or haplotypes seem to be involved in susceptibility to HPV infection and cervical neoplasia, probably by modulating immune response against HPV infection, and ultimately interfering in the establishment of productive persistent infections and cervical lesions. To date, the majority of HLA and cervical neoplasia studies have focused on the HLA Class II genes, since HLA II molecules are known to be responsible for the presentation of foreign antigens to the immune system. The strongest associations have been found for genes in the HLA class II region (Coleman et al, 1994; Evans et al, 2001; Maciag et al, 2000). In particular, the class II DQ allele shows evidence of allelic association with cervical neoplasia in HPV-positive patients. Three groups of alleles/haplotypes known to be associated with cervical neoplasia have been extensively studied and include (1) DQB1*03 alleles (including DQB1*0301, DQB1*0302, and DQB1*0303); (2) DRB1*1501 and DQB1*0602 alleles; and (3) DRB1*13 (consisting of DRB1*1301-/5 alleles) and DQB1*0603). Among these, DQB1*03 alleles, and DRB1 1501 and DQB1 0602 alleles appear to increase the risk of cervical disease (Hildesheim et al, 2002). The most consistent finding is that individuals with the DQB1 03 alleles have an increased risk of HPV infection and cervical cancer (Maciag et al, 2000; Odunsi et al, 1997), and this association was observed for preinvasive and invasive disease and was found to be valid regardless of ethnicity. Moreover, increased risks of cervical cancer and CIN were observed for the DRB115 allele and the related DRB1 1501-DQB1 0602 haplotype among Hispanic and Swedish patients (Apple et al, 1994; Sanjeevi et al, 1996). Conversely, evidence suggests that DRB1*13 and/or DQB1*0603 in German, American, and French populations (Breitburd et al, 1996; Hildesheim et al, 1998; Madeleine et al, 2002) are likely to protect against the development of cervical cancer

However, despite the relative consistency of findings supporting a role for HLA in cervical carcinogenesis, several inconsistencies remain. The reasons of these inconsistencies are attributable to variations in regional HPV types, HPV subtypes, ethnic HLA allele patterns, and differences in HLA typing techniques. It is also possible that HLA alleles reportedly associated with cervical cancer are located in genes that are in linkage disequilibrium with the actual gene or genes responsible for this additional risk. Variations in linkage would be expected between populations, and would be expected to result in the discrepancies described above. Finally, susceptibility may depend on interactions between immune response genes, and the specific alleles identified to date may represent only a portion of an extended haplotype that regulates immune response to HPV.

B. Single nucleotide polymorphisms (SNPs)

The p53 codon 72 polymorphism, which codes for either arginine (ARG) or proline (PRO), was first demonstrated by Storey et al. to be associated with cervical cancer and this has since been confirmed by numerous other investigators (Storey et al, 1998). The ARG/ARG genotype was found to confer an elevated risk for cervical cancer compared with the ARG/PRO genotype, by enhancing the binding and degradation of p53 by oncogenic HPV E6. Individuals with the homozygous arginine form were found to be seven times more susceptible to HPV-related carcinogenesis than heterozygotes (Storey et al, 1998). However, subsequent reports failed to corroborate these findings, and others were inconclusive. These observed discrepancies could be related to the ethnicities of the populations studied, as this polymorphism is known to differ by geographic region.

In a meta-analysis by Koushik and colleagues (2004), P53Arg homozygosity was not associated with an increased risk of cervical neoplasia (OR=1.1; 95% CI, 0.9 to 1.3) as compared with P53Pro homozygosity and P53Arg/Pro heterozygosity. This association was found to be statistically significant for invasive lesions but not for preinvasive lesions, and was also found valid to a lesser extent in squamous cell carcinoma (OR=1.5; 95% CI, 1.2 to 1.9) and adenocarcinoma (OR=1.7; 95% CI, 1.0 to 2.7). However, reports published after this meta-analysis continued to be contradictory. Positive associations have been reported between cervical cancer and P53Arg homozygosity in Chile (Ojeda et al, 2003) and Mexico (Sifuentes Alvarez et al, 2003), whereas no associations were found in Argentina (Abba et al, 2003), central Italy (Cenci et al, 2003), or in Korea (Kim et al, 2001).

C. Epigenetic changes

Mutations (both point mutations and deletions) are not the only way in which a tumor suppressor gene can be inactivated, and in recent years the importance of epigenetic changes in the establishment of the malignant phenotype has been illuminated. In malignancies, some tumor suppressor genes are not transcribed because their promoter regions are methylated. Common examples of tumor suppressor genes that are inactivated by promoter region hypermethylation are provided by INK4A locus and RASSF1A (Das et al, 2004).

Of the hypermethylation events studied in association with carcinogenesis, promoter CpG island hypermethylation has been frequently investigated in many human cancers, including cervical cancer (Esteller et al, 2001; Muller et al, 1998; Virmani et al, 2001; Yang et al, 2004). CpG islands are often associated with promoter regions, and hypermethylation of these regions, which is probably the best characterized epigenetic change, is associated with transcriptional silencing of the associated gene, and thus provides a DNA-based surrogate marker of expression status. Moreover, it has been increasingly recognized over the past 4–5 years that the CpG islands of a large number of genes, which are unmethylated in normal tissue, are methylated to varying extents in many human cancers, and that these methylations are a potential means of tumor suppressor gene inactivation (Kang et al, 2005).

At present, there is some evidence that increased ratesof hypermethylation of various genes may be associated withcervical cancer. Dong et al, (2001) showed that promoterhypermethylation of at least one of the genes p16, DAPK, MGMT,APC, HIC-1, and E-cadherin occurred in 79% of cervical cancertissues and in none of normal cervical tissues from 24 hysterectomyspecimens. Virmani et al, (2001) detected aberrant methylationof at least one of the genes p16, RARß, FHIT, GSTP1,MGMT, and hMLH1 in 14 of 19 cervical cancer tissue samples. In addition its implications in cervical tumorigenesis, DNA promoter hypermethylation are being investigated as a novel diagnostic target based on methylation-sensitivePCR techniques. Recently, Feng et al reported similar promoter methylation patternsin genes from exfoliated cell samples and correspondingbiopsy specimens. Furthermore, the frequency of hypermethylation increased statistically significantlywith increasing severity of neoplasia present in the cervicalbiopsy. (Feng et al, 2005).

V. Co-factors of cervical cancer

It has been well established that a persistent infection in combination with high-risk HPV is the main risk factor of cervical cancer. Although HPV infection is necessary for the genesis of cervical cancer and its precursors, HPV infection alone is by no means sufficient cause. Subclinical, clinical, and latent HPV infections are considered the most common sexually transmitted infections. Although age dependent, the prevalence of HPV infection among sexually active young women is in the range of 5-40% (Ho et al, 1998; IARC Working Group, 1995; Melkert et al, 1991). Further, most HPV infections are transient. It is estimated that newly diagnosed HPV infections will clear within 12–18 months in approximately 80% of women, as the humoral immune system is brought to bear on the virus. Even low-grade precancerous lesions do not always progress into high-grade lesions. Instead, a cytotoxic T cell response is elicited against HPV-infected keratinocytes in the majority of cases. This suggests that other factors are involved in the carcinogenesis of cervical cancer. Smoking, high parity, and the long-term use of oral contraceptives are considered proven co-factors. Others are being scrutinized by ongoing research. Figure 3 depicts a multifactorial model of cervical cancer etiology.

A. Smoking

Smoking has long been associated with cervical cancer risk after Winkelstein first proposed the hypothesis that smoking is a risk factor of cervical cancer (Winkelstein Jr, 1977). This hypothesis has been supported by subsequent epidemiological studies (Castellsague et al, 2003), although it has been found difficult to rule out residual confounders, chiefly arising from sexual habits known to be related to both smoking and cervical cancer.

Various mechanisms have been proposed to explain the association between smoking and cervical cancer. Tobacco is able to induce its carcinogenic effect in sites not directly exposed to cigarette smoke, as in pancreatic, kidney and bladder cancer (IARC Working Group, 1986). In the cervix, it is possible to detect nicotine derivatives like nicotinine and tobacco specific nitrosamines. In addition, DNA adducts and other evidence of genotoxic damage are detectable in exfoliated cervical cells (Szarewski et al, 1998). It has been shown that smoking affects the ability of the host to mount an effective local immune response against viral infections in the cervix, and smokers show reductions in the number of Langerhans cells and in other markers of immune function (Poppe et al, 1995). Another possibility concerns the systemic effect of smoking, whereby the metabolisms of female hormones are altered.

Despite the consistency of the association between smoking and cervical cancer after adjusting for sexual behavior, it is not generally agreed that confounding can be ruled out as an explanation for this finding, since sexual behavior is evidently associated with transmission. Now that HPV has been identified as the principal cause of cervical cancer, it should be possible to resolve controversies over smoking. However, relatively few smoking targeted studies have incorporated adjustment for HPV infection

A recent review of the relation between smoking and cervical cancer found consistent associations after adjusting for HPV-DNA or restricting analysis to HPV-positive women (Szarewski et al, 1998). These findings among HPV-positive women concur with subsequent studies (Deacon et al, 2000; Hildesheim et al, 2001; Plummer et al, 2003). Interestingly, in a multi-center case–control study of cervical adenocarcinoma, Lancey et al. reported a negative association between smoking and cervical adenocarcinoma (i.e., current smokers: OR = 0.6, 95% CI 0.3±1.1) and a marginal positive association between smoking and the risk of squamous cell carcinoma (e.g. current: OR = 1.6, 95% CI 0.9±2.9) (Lacey Jr et al, 2001).

Although a number of studies show that smoking is associated with an increased cervical cancer risk, further research using prospective designs that are well-controlled for HPV markers, confounding factors, and histologic types are needed to determine the nature of the relationship between smoking, HPV infection, and cervical cancer risk.

B. Sexually transmitted diseases

HSV-2 has been found to be carcinogenic in both in vitro and in vivo studies. Several possible mechanisms for the role of HSV-2 in cervical cancer have been suggested. It was hypothesized that HSV-2 and HPV may act synergistically with HSV-2 to initiate mutations and carcinogenesis in HPV-infected cervical cells (de Sanjose, 1994; Zur Hausen, 1982). However, because of a lack of consistency in detecting HSV-2 DNA in cervical cells, it has been postulated that a “hit-and-run” mechanism may play a role in the initiation of cervical cancer (Galloway et al, 1983).

Serologic studies showed a higher prevalence of HSV-Ab in women with cervical neoplasia than in controls (de Sanjose et al, 1994; Dillner et al, 1994). The results of a pooled analysis of case-control studies conducted by IARC support the role of HSV-2 as a cofactor of HPV infection in cervical cancer (Smith et al, 2002a). In this study, HSV-2 seropositivity was found to be significantly higher among women with invasive squamous cell carcinoma (44.4%) and adeno- or adenosquamous carcinoma (43.8%) than in control women (25.6%); this association was observed after adjusting for potential confounders.

A consistent but modest association between the presence of serum Ig G antibodies to C. trachomatis and cervical cancer has been reported in epidemiological studies (de Sanjose et al, 1994; Dillner et al, 1994). However, in other studies, infection with Chlamydia trachomatis was not found to be associated with the presence of HPV (Burger et al, 1996). In a IACR multicenter study, C. trachomatis seropositivity increased the risk for cervical cancer among HPV-positive women by 2.1-fold (Smith et al, 2002). In all, it appears that the lack of consistency shown by studies suggests that residual confounding due to HPV may have affected the finding of a positive association between cervical cancer and C. trachomatis.

Numerous studies have addressed the association between HIV and cervical neoplasia (Boyle et al, 1999), and the Center for Disease Control and Prevention included invasive cervical cancer in its definition of AIDS. Moreover, HIV-positive women have been reported to have higher rate of cervical abnormalities, larger lesions, and a higher recurrence rate than HIV-negative women (Jay et al, 2000; Conley et al, 2002). In addition HIV-positive women have been reported to have higher rates of HPV infection (40% to 95%) and CIN lesions (10% to 36%) than HIV-negative women (23% to 55% and 1% to 12%, respectively) (Ellerbrock et al, 2000; Moscicki et al, 2000; Ferenczy et al, 2003)

A meta-analysis by Mandelbaltt and colleagues concluded that HIV is a cofactor of HPV-related cervical carcinogenesis, and that this association seems to vary with immune function level (Mandelblatt et al, 1999). Although the biologic mechanism for this interaction is not well understood, it is explained as being due to the effect of HIV infection on the immune system and a molecular interaction between HIV and HPV.

C. Oral contraceptives

Steroid contraceptive hormones have been identified to be a cofactor of HPV-related cervical carcinogenesis in many, but not all, epidemiological studies. However, although some epidemiologic studies have produced inconsistent results, the majority of studies have found that prolonged used of these agents increases the risk of cervical cancer (Moreno et al, 2002; Castellsague et al, 2003).

Little data is available about the mechanisms by which OCs increase the risks of acquiring or progressing HPV infection to cervical cancer. Two possible mechanisms have been proposed, i.e., increased exposure of the transformation zone to potential carcinogens and increased cell proliferation and transcription. An increased incidence of cervical ectropion has been reported among OC users, and this would increase the likelihood of transformation zone exposure to HPV and other potential carcinogens. Moreover, the hypothesis concerning the stimulation of cell proliferation and HPV transcription by estrogens and progesterone is gaining support. Steroids are believed to bind to specific DNA sequences within transcriptional regulatory regions on HPV DNA, to either increase or suppress the transcriptions of various genes (de Villiers et al, 2004; Moodley et al, 2003).

Results from epidemiologic studies, in which HPV status was controlled for, demonstrate, in most cases, positive correlations between OC use and cervical cancer risk. Kruger-Kjaer et al reported a pattern of decreasing risks of ASCUS, LSIL and HSIL with years with OC use among HPV DNA positive women (Kruger-Kjaer et al, 1998). Recently, the IARC’s pooled analysis of eight case-control studies reported that the odds ratio of cervical cancer resulting from the use of oral contraceptives was 2.82 (95% CI 1.46–5.42) for 5–9 years, and 4.03 (2.09–8.02) for use for 10 years or longer (Moreno et al, 2002). In another recent review article, Smith et al. concluded that the relative risk of cervical cancer increases with oral contraceptive use duration (Smith et al, 2003). These findings are consistent for HPV positive women and after adjusting for HPV status. However, confounding must also be considered because women using contraception are more likely to be sexually active. Further, barrier methods of contraception have been shown to protect against cervical intraepithelial neoplasms and cervical cancer. Moreover, detection bias may also affect these results, because women using oral contraceptives have more frequent gynecologic visits than non- users, and therefore, precancerous lesions are more likely to be detected and treated.

VI. HPV vaccine

A. Prophylactic HPV vaccine

DNA-free virus-like particles (VLP) synthesized by the self-assembled viral particles of the main structural HPV proteins, L1 protein (or L1 and L2 protein), induce strong humoral responses from neutralizing antibodies. VLPs are thus the best candidate immunogens currently available for HPV vaccine trials. These VLPs are morphologically indistinguishable from the authentic virion, are non-infectious, and lack any oncogenic DNA. Several studies in animals have demonstrated that the parenteral injection of these VLPs, or even of the pentameric L1 capsomer, elicits high titers of serum-neutralizing antibodies and protection (Breitburd et al, 1995; Kirnbauer et al, 1996) .

As for human studies, early phase I/II clinical trials using HPV L1 VLP delivered intramuscularly have demonstrated the immunogenicity and safety of this vaccine (Evans et al, 2001; Harro et al, 2001). Importantly, Koutsky et al recently reported on a clinical trial of HPV 16 L1 VLPs, and indicated for the first time that a vaccine strategy can be implemented in humans to prevent HPV-16 infections and HPV-16–associated premalignant lesions (Koutsky et al, 2002). In another clinical trial reported by Harper et al, HPV-16, 18 L1 VLP vaccines proved 100% effective at preventing the acquisition of persistent HPV infection (Harper et al, 2004).

Several important issues require careful consideration before anti-HPV vaccines are made available for mass immunization programs. Humoral immunity to VLP-based vaccines is not only species specific but also type specific. Therefore, the number of HPV types to be included as immunogens is a key issue in HPV vaccine development, although single-type-specific VLP vaccines have produced encouraging results. Data from a recent overview of information collated from several case-control studies indicated that a pentavalent vaccine with VLPs of HPV types 16, 18, 45, 31, and 33 could potentially prevent 83% of all cervical carcinomas (Munoz et al, 2004). However, it is evident that the gains achieved by type coverage rapidly diminish for vaccines containing more than four types. A quadrivalent HPV VLP vaccine (types 6, 11, 16, and 18) produced by Merck is currently undergoing clinical trial; preliminary results show that the vaccine is well tolerated and generates adequate neutralizing antibody titers (Brown et al, 2001).

In addition to the type of HPVs covered by vaccines, the route of delivery is also an issue. Although VLP vaccination provides immunity from experimental inoculation, protection against the sexual transmission of HPV requires neutralizing antibodies acting at mucosal surfaces. The nasal instillation of VLPs was found to be efficient at generating specific antibodies, including IgG in serum and IgG and IgA in the mucosal secretions of mice (Balmelli et al, 1998). More recently, oral vaccination with HPV VLPs in mice was found to induce systemic virus-neutralizing antibodies (Rose et al, 1999).

Other important issues must also be resolved, such as, age at time of administration, booster shot timing, and whether or not to vaccinate men. Finally, given that cervical cancer does not develop in the vast majority of women infected with HPV, economic benefit should also be taken into consideration. In addition, because cervical cancer remains important public health problem in low-income countries, the cost of the vaccine for developing countries should also be consideration.

B. Therapeutic HPV vaccine

Although vaccination with prophylactic HPV vaccines can generate high titers of serum-neutralizing antibodies in animals and humans, this form of immunization may not be able to generate the therapeutic effects required to counter established or breakthrough HPV infections that have escaped antibody-mediated neutralization. Preexisting HPV infection is highly prevalent and is responsible for considerable morbidity and mortality.

The life cycle of a HPV infection is characteristically intracellular, noncytopathic, and nonlytic. Therefore, the goal of therapeutic vaccination is to induce specific cell-mediated immunity targeting preexisting lesions or even malignant tumors. In the case of cervical cancer, viral peptides derived from high-risk HPV oncoproteins are tumor-specific antigens, because viral genes are selectively expressed during the malignant progressions of virally induced neoplastic lesions. As a result early viral antigens (i.e., E1, E2, E5, E6, and E7) could be candidate targets for therapeutic vaccine antigens. However, most HPV-associated cancers only express E6 and E7, and E5 shows limited immunogenicity, and thus has not been extensively studied as a vaccine antigen. Likewise, E4 and the L1 and L2 capsid proteins are unlikely to be suitable targets for therapeutic vaccine development, because these proteins are not detectably expressed in the basal epithelial cells of benign lesions or in the abnormal proliferative cells of premalignant and malignant lesions (Stoler et al, 1992). Furthermore, because E6 and E7 are required for the induction and maintenance of the malignant phenotype of cancer cells, cervical cancer cells are unlikely to evade an immune response through antigen loss. Thus, the majority of investigations on therapeutic vaccines are directed toward E6 and E7 antigens.

The various categories of therapeutic vaccines are; vector-based, peptide-based, protein-based, DNA-based, chimeric VLP-based, and cell-based. However, most studies have focused on E7, because it is more abundantly expressed and better characterized immunologically, and because its sequence is more conserved than that of E6 (Zehbe et al, 1998). A summary of prophylactic and therapeutic vaccines currently being studied is presented in Table 2.

VII. Future prospects

During the past few decades, the molecular mechanisms underlying the development and progression of HPV-associated cervical neoplasms have been extensively studied, and as a result a new appreciation for these mechanisms of cervical neoplasia development has emerged. Moreover, it has been established that HPV oncogenes are not only indispensable for malignant transformation, but that they are also important functional regulators of the various key genes involved in cervical carcinogenesis.

However, although various molecular interrelationships involved in HPV-associated-cervical carcinogenesis have been identified, the essential molecular genetic pathway remains to be elucidated. Future studies should be directed at determining the following: (1) the roles of other HPV early proteins (such as E1, E2 and E4); (2) the oncogenicity of specific HPV variants, (3) the host control mechanism against HPV infection; (4) the mechanism of apoptosis modulation by HPV oncoprotein; (5) the interaction between HPV oncogene and co-factors; (6) the nature of cervical cancer risk genes; (7) the correlation between viral load and severity of disease; (8) the identities of additional vaccine targets, such as L1 and L2 and (9) those of the biomarkers of cervical neoplasm progression, and finally (9) inexpensive, low-technology HPV diagnostics should be developed.

Cervical cancer is a multifactorial and dynamic event in which numerous alterations contribute to disease development. Thus it is hoped that a better understanding of the pathogenic roles of HPV oncoprotein will help advance mechanism-based screening tools and therapies for the prevention and treatment of cervical cancer, and provide an insight of the fundamental rules of cervical carcinogenesis.

Acknowledgement

Korean Health 21 R&D Project, Korean Ministry of Health &Welfare; Grant number: 0412-CR01-0704-0001.

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Early region	Protein functions
E1	Unwinds the DNA strands working with E2 protein Modulate the transcription activity of the E2 protein
E2	Enables E1 protein to bind to the viral origin of replication located within the LCR Encodes a LCR-binding protein that regulates transcription of the early region
E4	Encodes a protein that interacts with cytokeratin Expressed in later stages of infection, when complete virions are being assembled
E5	Augment cellular proliferation and DNA synthesis in a context of cell membrane receptors, such as EGF and PDGF Induces an increase in mitogen-activated protein kinase activity
E6	Binds to p53 and targets it for rapid degradation via a cellular ubiquitin ligase Induces telomerase activation
E7	Binds to the hypophosphorylated Rb proteins and liberate E2F, which results in S phase entry Interacts with inhibitors of cyclin dependent kinases Induces abnormal centrosome duplication resulting in aneuploidy

Target	Vaccine	Phase	Results	Company
HPV 16 L1 VLP HPV 18 L1 VLP	Cervarix	Phase II	100% protection against persistent HPV 16/ 18 infection, well tolerated	Glaxo-Smith Kline
HPV 6 L1 VLP HPV 11 L1 VLP HPV 16 L1 VLP HPV 18 L1 VLP	Quadrivalent vaccine	Phase III	90% decrease in Combined incidence of persistent infection or disease with HPV 6, 11, 16, or 18, well tolerated	Merck
HPV 16 E6, 7 HPV 18 E6, 7	TA-HPV-Vaccina virus encoding	Phase I, II	Clinical responses in women with long-standing high-risk HPV positive VAIN or VIN, well tolerated	Xenova
HPV protein	TA-CIN/TA-HPV	Phase II	Induction of HPV 16-specific T-cell and/or serological responses in HPV positive VIN patients, clinical response.	Xenova
HPV-16 E7	HSP-E7 – protein and/or peptide based vaccine	Phase II	CD-8-dependent and CD-4-independent regression of HPV-16 E-7 expressing tumors in mice Currently in human trials	StressGen
HPV DNA	ZYC101	Phase I, II	Some responses observed in cervical dysplasia patients, well tolerated	Zycos

Review Article

Received: 31 May 2005; Accepted: 22 June 2005; electronically published: July 2005

References