Key words: CD95/TRAIL/TNF-a pathway; immunological escape; antiviral host defense; cervical cancer; HDAC; sodium butyrate; p53; pRb, E2F.

Abstract

The oncogenic potential of “high risk” human papillomaviruses can be mainly attributed to two small proteins called E6 and E7. Even these oncoproteins have a low molecular size, they are highly promiscuous and are capable to interact with a whole variety of host cellular regulator proteins to elicit cellular immortalization and ultimately complete malignant transformation. To avoid reiterations in summarizing the biochemical and molecular biological properties of E6/E7 in terms of their influence on cell cycle control, the present review is mainly an attempt to describe some regulatory principles by which HPV oncoproteins can interfere with apoptosis in order to escape immunological surveillance during progression to cervical cancer. The models derived from these basic cellular and molecular studies are relevant to our understanding of HPV-induced carcinogenesis. Conversely, experimental procedures aimed at relieving apoptosis resistance, can facilitate the eradication of immunologically suspicious cells and may prevent the accumulation of cervical intraepithelial cell abnormalities in future prophylactic or therapeutic approaches.

INTRODUCTION

Specific types of human papillomavirus (HPV) – mainly type 16 and 18 (“high-risk“) – have been identified as causative agents of at least 90 % of cancer of the cervix uteri and are also etiologically linked to more than 50 % of other anogenital cancers (for review, see Ref. [1]). Both HPV types possess immortalizing and transforming activity for human cells (for reviews, see Refs. [2, 3]), a biological property, which is genetically confined to two small open reading frames (ORF) encoding the proteins E6 and E7 (Fig. 1A). To act as oncogenes, however, E6 and E7 have to be uncoupled from their normal function during the regular permissive life cycle [4, 5]. The major process, which is creating such a situation is the physical linkage of the virus with the host cell DNA. In premalignant lesions, HPVs regularly persist as autonomous replicating episomes, while the viral DNA is found to be integrated in a monoclonal fashion in the majority of cervical cancers [6, 7]. In fact, integration provides a selection advantage towards progression, because it guarantees the continuous expression of E6/E7 without loosing the viral templates due to extrachromosomal segregation or encapsidation. Although there is ample evidence that both oncoproteins are necessary for the maintenance of the transformed phenotype [8, 9], their expression alone is not sufficient to immortalize and transform human cells by a single hit mechanism. HPV-induced anogenital cancer can therefore be considered as a multi-step process (for review, see Refs. [10, 11]), which has at least four consequences for the host cell (Fig. 1B):

a) integration leads to the disruption of the early coding region often located between the E1 and E2 ORF, which relieves the negative regulatory effect of E2 on its own promoter/enhancer region and concomitantly on E6/E7 expression (for review, see Ref. [12]). b) due to integration, virus-cell-fusion transcripts are generated, whereby the own poly-A signal is lost. These E6/E7 mRNA species usually acquire a higher stability, which in turn raises the amount of intracellular viral oncogenes [13]. Moreover, random linkage with the host genome can modulate E6/E7 expression either by increasing or decreasing the initiation rates of viral transcription through adjacent 5´-cellular regulatory sequences (= position effects) [14, 15]. c) viral integration during persistence can directly inactivate or dys-regulate cell cycle controlling genes via insertional mutagenesis. Since this event has to take place in a homozygous fashion, its probability is rather low, but not without a precedence [16]. d) continuous viral oncogene expression affects cellular signaling or/and particular cellular transcription factors both on transcriptional and translational level, whose functional abrogation ultimately favors host cell transformation [10].

Nevertheless, it should be emphasized that not all persistent HPV infections culminate in the development of cancer. There is also experimental support for the existence of intra-and extracellular surveillance strategies which prevent the accumulation of malignant cells either by ablating oncogene expression and function [11] or by elimination of infected cells via apoptosis.

THE ROLE OF E6/E7 IN THE REGULATION OF APOPTOSIS

Escape from immunological control can be favored by the following mechanisms: a) through down-regulation of MHC class I expression, loss of transporter protein TAP-1 expression and concomitantly the ability to present foreign antigens [17-19]. b) disturbance of cell-cell communication between HPV-infected keratinocytes with immunological effector cells as a result of dysfunctional chemokine/cytokine expression (for review, see Ref. [20], [21]. c) and by ignoring apoptotic stimuli accomplished by the CD95/TRAIL/TNF-a signal transduction pathway (for review, see Refs. [22, 23]).

By definition, apoptosis is a genetically determined program which finally leads to the induction of caspase-activated deoxyribonuclease [24]. Consequently, not only the high molecular DNA becomes cleaved, but also replicating or persisting viruses not yet packaged into mature viral capsids. For this reason, apoptosis of virus-infected cells can be regarded as an altruistic mode to limit the spread of progeny virions into the host organism. Besides other routes of immunological control, evasion from apoptosis in terms of changing the numerical equilibrium between cell loss (= through immunological removal) in favor to cell gain (= through uncontrolled proliferation) may conduct to serious clinical manifestations lastly ending up in tumor formation [23].

During the last few years, there are various reports in the papillomavirus field providing examples of how viral oncogenes of certain HPVs can interfere with the ability to undergo programmed cell death. In fact, using the “TUNEL” technique to measure the degree of DNA fragmentation directly within primary tissue sections [25], premalignant HPV-positive lesions exhibit reduced apoptotic indices of undifferentiated basaloid-like cells, which significantly correlates with the probability of progression to squamous cell carcinomas [26]. Basically, apoptosis can be induced by a wide range of different cell-intrinsic signals such as nutrient depletion [27], hypoxia or chemotherapeutic drugs (for reviews, see Refs. [28, 29]). The most physiological pathway to switch on the death machinery, however, is the engagement of natural apoptosis-inducing ligands (CD95-L / TNF-related apoptosis-inducing ligand (TRAIL) / TNF-a) to their corresponding receptors at the surface [22, 23]. The suicide receptors belong to a superfamily of transmembrane proteins which are parts of the TNF-receptor (TNF-R) superfamily (for review, see Ref. [30]). After ligand binding, receptor oligomerization is followed by a recruitment of the FADD protein (Fas-associated-death domain) and procaspase 8 to build up a death-inducing signaling complex (DISC) [31] (Fig. 2A). DISC formation induces sequential proteolytic cleavage of downstream executioner caspases (for review, see Ref. [32]) as well as distinct morphological features such as cell shrinkage, nuclear condensation and internucleosomal DNA fragmentation [33]. In the case of CD95-mediated apoptosis, two different pathways have been unraveled, which are schematically outlined in Fig. 2B: in type I cells, apoptosis is initiated by a strong DISC formation leading to rapid activation of initiator caspase-8 and caspase-3. In type II cells, DISC formation is diminished and temporarily delayed, whereby the executioner procaspase-3/9 are activated by disruption of the mitochondrial membrane and the release of apoptogenic factors such as cytochrome c. Type II cell death can be inhibited by over-expression of Bcl-2 or Bcl-XL [34, 35]. CD95/TNF-a-mediated apoptosis is negatively regulated by FLICE/caspase-8-inhibitory proteins (FLIPs), which comprise both cellular and viral members engaged in transformation and viral persistence [35]. These proteins can act as decoys by competing with procaspase 8, because they are similar in structure but lack essential catalytic domains for proteolysis [35].

Humanpathogenic DNA viruses have developed efficient strategies to modulate apoptotic response upon infection (for review, see Ref. [36]). Adenovirus-infected cells for example, can overcome the antiviral host defense and promote their own survival by forcing the degradation of the CD95-receptor [37]. In the case of HPV, there is also experimental evidence that the HPV 16 E5 protein can reduce the amount of CD95 receptor at the cell surface [38]. However, since E5 often becomes deleted in cervical carcinoma cells [12], the physiological relevance of decreased CD95 surface expression is probably restricted to the natural life cycle, where temporal blockage of apoptosis protects replicative intermediates and immature virions from caspase-induced DNA cleavage. This notion is supported by the finding that CD95/TNF receptor 1 (TNF R1) surface expression is not quantitatively altered in immortalized/malignant HPV-positive cells when compared with primary human keratinocytes [39].

In contrast, HPV 16/18 E6/E7 oncoproteins can efficiently modulate other branches of the apoptotic pathway, although the outcome evidently depend on the cellular model system, the viral type and in particular, on the nature of the stimulus. For example, HPV 16 E6 sensitize immortalized human mammary epithelial cells to apoptosis in the presence of tamoxifen or DNA damaging agents [40], but protects lens fiber cells during the natural fiber differentiation in a both p53-dependent and independent fashion [41]. On the other hand, E7-immortalized human fibroblasts were almost resistant against TNF-*/cycloheximide-mediated cell death [42], but not against the TNF-related apoptosis-inducing ligand (TRAIL) in keratinocytes [43]. Both oncogenes, however, augment apoptosis of human epithelial cells when exposed to hypoxic conditions [44], while HPV16-positive cervical carcinoma cells emerged as less prone to CD95-mediated cell killing [45].

Since E6 and E7 are profoundly changing the expression profile of an infected cell [46], it is unlikely that all these effects are carried out simultaneously. In the case of viral persistence, one has to postulate that oncoprotein activity has a well defined temporal and spatial order, which can be regulated by viral promoter efficiency [12], fine-tuning of E6/E7 cooperativity [47], post-translational modification [48] and intracellular compartimentation [49]. One way to dissect at least the inherent collaborative effect of both oncogenes on the apoptosis machinery is their separate expression in appropriate model systems. In the course of these, but also in other experiments it became clear that even there exist different modes of cell death, the most prominent function of the E6 protein is the proteolytic inactivation of certain proapoptotic factors such as p53 [50], Bak [51], Bax [52] or c-Myc [53] through the ubiquitin-proteasome pathway. Moreover, since E6 ORF transcription is not only controlled at the level of initiation of transcription [12], but also by differential splicing of the polycistronic RNA, full length E6 and a truncated version E6, referred as E6* can be translated [54]. The intracellular ratio between these two proteins in turn may have important implications on the commitment to undergo programmed cell death both in immortalized/malignant cells and terminally differentiating keratinocytes. This can be mainly attributed to the ability of E6* to interact with E6 and the ubiquitin-protein ligase E6-AP [55], thereby inhibiting proteasome-mediated proteolysis and favoring apoptosis [47]. Hence, quantitative changes of the E6/E6* stoichometry, especially during the productive cycle, may maintain the half-life of survival proteins like Bak and Bax, whose expression become normally enhanced in suprabasal layers during epithelial differentiation [56].

Nonetheless, inactivation of p53 itself seems to represent a key regulatory event in CD95-induced apoptosis. As shown recently, application of CD95 ligand only renders E7-immortalized cells to extensive apoptosis, while E6- and E6/E7-expressing keratinocytes were resistant [39]. Notably, the latter can be fully sensitized to ligand induced cell death by short-term blockage of proteasomal degradation, which was preceded by the re-expression of p53 and c-Myc, but not of other half-life controlled pro-apoptotic proteins such as Bax, despite the gene is considered to be p53 responsive [57]. CD95-induced apoptosis was neither accomplished by proteasome inhibition alone, nor in cells lacking functional p53, even though c-Myc as potential survival factor [58] was induced. This contrasts an observation made in rodent cells, where Rat1 fibroblasts were not susceptible to CD95 cell death except c-Myc was expressed [59]. Remarkably, the mere elevation of p53 was insufficient to cause apoptosis in E6-and E6/E7 cells under these condition, unless the corresponding CD95 ligand was provided [39]. This is consistent with the idea that receptor activation triggers additional modifications of p53. In fact, diverse extracellular stimuli can cause distinct phosphorylation of p53 in a post-translational fashion, activating alternative sets of genes either engaged in cell cycle control or in apoptosis [60].
,br> There are also other functional pathways by which E6 can exert an anti-apoptotic function: delivery of E6, for instance, protects mouse and human cells from TNF-a mediated cell death in a p53-independent manner [61]. Although TNF-a, in contrast to CD95, may also induces additional cytoprotective branches such as activating the NF-kB or MAP-kinase pathway [62], both mechanisms are not mutually exclusive [63]. While HPV 16 E6 does not decrease the level of the TNF-receptor (TNF R1), inhibition can be attributed to the inability of the TNF R1 intracellular death domain to interact with FADD in the presence of E6. Resistance is exerted through physical binding to the C-terminal part of TNF R1, thus preventing the activation of procaspase 8 and the downstream procaspase 3, both necessary for successful transmission of the apoptogenic response [61]. The notion that HPV 16 E6 interferes with TNF-a signal transduction is consistent with a previous report [45], in which malignant cells harboring different types of HPV were monitored towards apoptotic signals such as CD95 and TNF-a. In this study, HPV18-positive cell lines were found to be highly sensitive to both ligands after co-exposure with cycloheximide, while HPV16-positive cervical carcinoma cells were refractory under equivalent conditions. Remarkably, CD95/TNF-a resistance can be suppressed after somatic cell hybrid formation between resistant and sensitive cells, clearly indicating that the latter phenotype is a dominant trait. Similar to the situation described above, HPV 16 positive (CD95 / TNF-a resistant) cells apparently failed to recruit FADD and procaspase 8 through the death effector domain. It is therefore reasonable to assume that the failure to form a functional DISC represents one way by which HPV16 positive cells could circumvent apoptotic signals during multi-step progression to cervical cancer [45]. Consequently, the development of small molecules capable to interfere with E6 and its interaction with host cell regulatory proteins [64] can be considered as a successful up-coming therapeutical strategy to treat dysplastic and neoplastic lesions.

“High-risk” E7 oncoproteins have also pleiotropic effects on the host cell. Expression of E7 is sufficient to immortalize primary human keratinocytes, although the immortalizing capacity is more efficient in the presence of E6 as cooperating oncogene [65]. E7 can uncouple differentiation and proliferation by promoting cell cycle progression. This property is mainly executed through the disruption of the repressor complex formed between the retinoblastoma protein pRb and E2F, a transcription factor, which trans-activates genes involved in DNA synthesis [2]. Moreover, E7 can also interfere with the activity of the cyclin-dependent kinase inhibitors p21CIP1 and p27KIP1 to override normal G1 checkpoint control [66, 67]. Since the metabolic half-life of pRb is inherently controlled by E7 [68], its reduced intracellular availability may also diminish the anti-apoptotic activity of pRb [69], thereby contributing to E7-prone apoptosis [70].

Similar to the situation described for CD95 sensitivity [39], enhanced commitment to undergo apoptosis can be also detected in E7-expressing immortalized human keratinocytes, when exposed to the inflammatory mediators TNF-a [71] or TRAIL [43], but only in conjunction with cycloheximide (CHX). Inhibitors such as CHX or actinomycin D are known to sensitize to ligand mediated apoptosis by blocking the de novo synthesis of short-lived cytoprotective proteins [72]. Alternatively, cycloheximide may prevent NF-kB-mediated up-regulation of c-FLIP, alleviating the anti-apoptotic effect of NF-kB activation induced upon ligand application [73]. Depending on the cell type, E7 can also attenuate the apoptotic response. When delivered in normal human fibroblasts, E7 decreases the activation of procaspase-8 which protects against TNF-a / CHX induced cell death [78].

On the other hand, expression of E7 alone apparently sensitizes keratinocytes to undergo apoptosis in an inherent fashion, not only by death ligands, such as TRAIL [43] or CD95L [39], but also through modulation of the equilibrium between histone deacetylases (HDAC) with antagonizing histone acetylases (HAT), known to regulate the switch from eu- to heterochromatin [75]. Not only cellular transcription factors can physically interact with HATs and HDACs, but also viral proteins. HPV 16 E6 is abrogating the co-stimulatory function of CBP and p300 [76], while E7 can indirectly bind to HDAC via the bridge-protein Mi2b [77].

In a recent study it has been demonstrated that HDAC inhibition can induce growth arrest and subsequently strong apoptosis in E7-expressing cells, but not when E6 was present alone [78, 79]. The key regulatory event under these conditions seems to be the degradation of the anti-apoptotic protein pRb [80] without any reduction of the transcription factor E2F-1, whose unscheduled intracellular accumulation favors programmed cell death (for review, see Ref. [81]). Disappearance of pRb after HDAC addition could be clearly attributed to a post-translational event, because the steady-state level of the corresponding mRNA was maintained. In cells containing E6 as viral oncogene, only hypophosphorylation could be discerned, clearly indicating that the fate of pRb was affected by E7 in a dominant mode. Improper release of E2F-1 during pRb degradation, jointly with a block in G1 to S transition of the cell cycle, provides a prerequisite for the classical “conflict model” in which cells were committed to undergo apoptosis [82]. Different apoptosis sensitivity of E6-versus E7-and E6/E7-expressing cells could be explained that E2F becomes acetylated by co-activators such as p300/CBP and p/CAF (p300/CPB-associated factor), particular adaptor molecules, which have intrinsic HAT activity [83]. Acetylation in turn results in increased DNA-binding affinity, trans-activation and prolonged half-life [84]. However, when bound to pRb, this effect can be efficiently reversed by pRb-associated HDAC 1 activity [85]. Since the steady-state level of p53 was not affected under these experimental conditions, forced degradation of pRb in E7-positive cells after HDAC inhibition may favor the induction of E2F-responsive pro-apoptotic genes such as caspase 3 [86] or the p53 homologue p73 [87]. Conversely, in E6-expressing keratinocytes, HDAC inhibitors may certainly also block deacetylation, but hypophosphorylated pRb prevents the release of E2F, while associated p/CAF HAT as a functional counterpart is still active. The fact that inhibition of histone deacetylation can bypass the transforming potential of "high-risk" HPV oncoproteins by inducing apoptosis may have important implications for the treatment of cervical cancer.

Acknowledgements
The authors thank Prof. Harald zur Hausen for critical reading of the manuscript and Dr. Luis Jave Suarez for the graphical design.

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Figure 1A. Genome organization of HPV16. The locations of the open reading frames encoding the early (E1-E7) and late (L1, L2) proteins as well as the long control region (LCR) are indicated. >
Figure 1B. Consequences of viral integration on HPV-specific gene expression.

Figure 2A.CD95-mediated formation of the "death-inducing signaling complex" (“DISC”).

Figure 2B.Type I/II pathway of apoptosis. (modified according to [34, 58]).

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