Transition to an irreversible state of senescence in HeLa cells arrested by repression of HPV E6 and E7 genes

Kim H.S., Song M., Kwak I.H., Park T.J., Lim I.K.

The mechanism of senescence-associated cytoplasmic induction of p-Erk1/2 (SA-p-Erk1/2) proteins in human diploid fibroblasts was investigated. p-Erk1/2 proteins were efficiently dephosphorylated in vitro by protein phosphatases 1 and 2A (PP1/2A) and MAPK phosphatase 3 (MKP3). Specific activity of PP1/2A and MKP3 activity significantly decreased during cellular senescence, whereas their protein expression levels did not. To investigate possible mechanism of phosphatase inactivation, we measured reactive oxygen species (ROS) generation by fluorescence-activated cell sorting analysis and found it was much higher in mid-old cells than the young cells. Treating the young cells once with 1 mm H2O2 remarkably induced p-Erk1/2 expression; however, it was transient unless repeatedly treated until 72 h. Multiple treatment of the cells with 0.2 mm H2O2 significantly duplicated inactivation of PP1/2A; however, thiol-specific reagents could reverse the PP1/2A activities, suggesting the oxidation of cysteine molecule in PP1/2A by the increased ROS. When the cells were pretreated with 10 mm N-acetyl-l-cysteine for 1 h, Erk1/2 activation was completely blocked. To elucidate which cysteine residue and/or metal ion in PP1/2A was modified by H2O2, electrospray ionization-tandem mass spectrometry analyses were performed with purified PP1C-α and found Cys62-SO3H and Cys105-SO3H, implicating the tertiary structure perturbation. H2O2 inhibited purified PP1C-α activity by both oxidation of Cys residues and metal ion(s), evidenced by dithiothreitol and ascorbate-restoration assay. In summary, SA-p-Erk1/2 was most likely due to the oxidation of PP1/2A, which resulted from the continuous exposure of the cells to vast amounts of ROS generated during cellular senescence by oxidation of Cys62 and Cys105 in PP1C-α and metal ion(s).

DeFilippis R.A., Goodwin E.C., Wu L., DiMaio D.

ABSTRACT Cervical cancer cells express high-risk human papillomavirus (HPV) E6 and E7 proteins, and repression of HPV gene expression causes the cells to cease proliferation and undergo senescence. However, it is not known whether both HPV proteins are required to maintain the proliferative state of cervical cancer cells, or whether mutations that accumulate during carcinogenesis eliminate the need for one or the other of them. To address these questions, we used the bovine papillomavirus E2 protein to repress the expression of either the E6 protein or the E7 protein encoded by integrated HPV18 DNA in HeLa cervical carcinoma cells. Repression of the E7 protein activated the Rb pathway but not the p53 pathway and triggered senescence, whereas repression of the E6 protein activated the p53 pathway but not the Rb pathway and triggered both senescence and apoptosis. Telomerase activity, cyclin-dependent kinase activity, and expression of c-myc were markedly inhibited by repression of either E6 or E7. These results demonstrate that continuous expression of both the E6 and the E7 protein is required for optimal proliferation of cervical carcinoma cells and that the two viral proteins exert distinct effects on cell survival and proliferation. Therefore, strategies that inhibit the expression or activity of either viral protein are likely to inhibit the growth of HPV-associated cancers.

Jeon J., Cho S., Kim C., Shin D., Kwon J., Choi K., Kim I.

Human papillomavirus E7 (HPV E7) is a viral oncoprotein that plays an important role in cervical carcinogenesis through binding with retinoblastoma protein (Rb). Inactivation of Rb by E7 is necessary but not sufficient for cellular transformation, suggesting other protein-protein interactions are required for E7-mediated cellular transformation aside from the interaction with Rb. However, studies on the oncogenic function of HPV E7 have been limited by its poor immunoreactivity. In this report, we show that the fixation of purified recombinant HPV E7 on blotted nitrocellulose membrane with glutaldehyde markedly enhanced the immunoreactivity of HPV E7 protein. Using HeLa and Caski cell line which are infected with HPV 18 and HPV 16, respectively, we demonstrated that native HPV E7 proteins also could be detected by this method. These results therefore can provide the experimental conditions for detection of HPV E7 proteins with greater sensitivity and may help to analyze E7 functions.

Hwang E.S.

Studies on the replicative senescence and premature senescence induced by various stresses in normal somatic cells have provided important clues on the role of telomere shortening and mechanisms involved in aging processes and carcinogenesis. Recent work revealed that cancer cells also are induced to undergo replicative senescence state via telomere shortening as well as to enter a senescence-like state by the activation of cell cycle inhibitory pathways. Although less relevant in terms of aging physiology, studies on these phenomena in cancer cells have yielded important information on telomerase regulation and the roles of tumor suppressors in senescence and immortalization, and are expected to generate valuable anti-cancer strategies. Several features of the phenotypes specific for the senescent and senescence-like states induced in cancer cells are discussed.

Lee C.J., Suh E.J., Kang H.T., Im J.S., Um S.J., Park J.S., Hwang E.S.

The E6 and E7 oncoproteins of human papillomavirus (HPV) play a major role in the development of cervical carcinoma. In this study, a recombinant adenovirus that expresses the bovine papillomavirus (BPV) E2, which has been shown to inhibit HPV early gene expression, was delivered to two HPV-immortalized cell lines as well as CaSki, a cervical carcinoma cell line. We tested whether the carcinoma and the immortal cells were equally affected by the expression of BPV E2. In all cell lines, BPV E2-mediated inhibition of HPV E6/E7 expression caused a dramatic suppression of cell growth, being preceded by the activation of the p53-Rb growth-inhibitory pathway, and a decrease in hTERT mRNA expression and telomerase activity. This suggests that the HPV E6 and E7 proteins are required not only for induction of the proliferative phenotype and telomerase activity, but also for their maintenance. In both the carcinoma and the immortal lines, the number of cells with enlarged cytoplasm and senescence-associated beta-galactosidase activity, which are markers for cellular senescence, was significantly increased. These results suggest that a senescence program exists in cells immortalized by HPV DNA as well as in cervical carcinoma cells.

Hwang S.G., Lee D., Kim J., Seo T., Choe J.

Chang B., Swift M.E., Shen M., Fang J., Broude E.V., Roninson I.B.

Treatment with chemotherapy or radiation is not invariably cytotoxic to all tumor cells. Some of the cells that survive treatment recover and resume proliferation, whereas others undergo permanent growth arrest. To understand the nature of treatment-induced terminal growth arrest, colon carcinoma cells were exposed to doxorubicin, and surviving cells were separated into proliferating and growth-arrested populations. Only growth-arrested cells displayed phenotypic markers of cell senescence and failed to form colonies. Gene expression was compared between senescent and proliferating fractions of drug-treated cells by using cDNA microarray hybridization and reverse transcription–PCR. Drug-induced senescence was associated with inhibition of genes involved in cell proliferation and with coinduction of multiple intracellular and secreted growth inhibitors. Several tumor suppressors and other genes that are down-regulated in carcinogenesis were up-regulated in senescent tumor cells. Induction of most growth inhibitors was delayed but not abolished in cells with homozygous knockout of p53, in agreement with only limited p53 dependence of drug-induced terminal growth arrest. On the other hand, senescent cells overexpressed secreted proteins with antiapoptotic, mitogenic, and angiogenic activities, suggesting that drug-induced senescence is associated with paracrine tumor-promoting effects. About one-third of the genes up-regulated in senescent cells and almost all of the down-regulated genes showed decreased or delayed changes in p21 Waf1/Cip1/Sdi1 -deficient cells, indicating that p21 is a major mediator of the effects of p53 on gene expression. Elucidation of molecular changes in tumor cells that undergo drug-induced senescence suggests potential strategies for diagnostics and therapeutic modulation of this antiproliferative response in cancer treatment.

Damm K.

Telomerase, the ribonucleoprotein enzyme maintaining the telomeres of eukaryotic chromosomes, is active in most human cancers and in germline cells but, with few exceptions, not in normal human somatic tissues. Telomere maintenance is essential to the replicative potential of malignant cells and the inhibition of telomerase can lead to telomere shortening and cessation of unrestrained proliferation. We describe novel chemical compounds which selectively inhibit telomerase in vitro and in vivo. Treatment of cancer cells with these inhibitors leads to progressive telomere shortening, with no acute cytotoxicity, but a proliferation arrest after a characteristic lag period with hallmarks of senescence, including morphological, mitotic and chromosomal aberrations and altered patterns of gene expression. Telomerase inhibition and telomere shortening also result in a marked reduction of the tumorigenic potential of drug-treated tumour cells in a mouse xenograft model. This model was also used to demonstrate in vivo efficacy with no adverse side effects and uncomplicated oral administration of the inhibitor. These findings indicate that potent and selective, non-nucleosidic telomerase inhibitors can be designed as novel cancer treatment modalities.

Tresini M., Lorenzini A., Frisoni L., Allen R.G., Cristofalo V.J.

Replicative senescence is characterized by numerous phenotypic alterations including the loss of proliferative capacity in response to mitogens and numerous changes in gene expression including impaired serum inducibility of the immediate-early genes c-fos and erg-1. Transcription of c-fos in response to mitogens depends on the activation of a multiprotein complex formed on the c-fos serum response element (SRE), which includes the transcription factors SRF (serum response factor) and TCF (ternary complex factor). Our data indicate that at least two defects are responsible for the decreased c-fos transcription in senescent cells, one caused by diminished DNA binding activity of the SRF and another resulting from impaired activation of the TCF, Elk-1. In nuclei isolated from serum stimulated senescent cells the activating phosphorylation of p62(TCF)/Elk-1, which is catalyzed by the members of the extracellular-regulated kinase (ERK) family was strikingly diminished and correlated with a decrease in the abundance of activated ERK proteins. In contrast, in total cell lysates ERK phosphorylation and ERK activity (normalized to total protein) reached similar levels following stimulation of early- and late-passage cells. Interestingly, senescent cells consistently exhibited higher ERK protein abundance. Thus, the proportion of phosphorylated (active) ERK molecules in stimulated senescent cells was lower than in early passage cells. The accumulation of unphosphorylated ERK molecules in senescent cells correlated with the diminished abundance of phosphorylated (active) MEK. These data indicate that in senescent cells there is a general dysregulation in the ERK signaling pathway, which results in the accumulation of inactive ERK molecules, decreased abundance of active ERK in the nucleus of senescent cells, and subsequent lack of activation of the transcription factor TCF(Elk-1). These impairments, together with the impaired DNA binding activity of SRF, could potentially account for the lack of c-fos expression in senescent cells and for multiple other molecular changes dependent upon this pathway.

Jung M., Yun J., Chae H., Kim J., Kim S., Choi T., Shin D.Y.

Recent studies have identified two p53 homologues, p63 and p73. They activate p53-responsive promoters and induce apoptosis when overexpressed in certain human tumors. Here, we report that p63, like p53 and p73, induces replicative senescence when expressed in a tetracycline-regulated manner in EJ cells lacking a functional p53. In addition to transcription activation of p53-responsive genes, we found that p63 and p73 repress transcription of the cdk1 and cyclin B genes, both of which are irreversibly repressed in senescent human fibroblast. In transient transfection assay, p63 and p73 repress the cdk1 promoter regardless of the presence of a dominant negative mutant form of p53. Furthermore, we found that DNA binding activity of NF-Y transcription factor, which is essential for transcription of the cdk1 and cyclin B genes and inactivated in senescent fibroblast, is significantly decreased by expression of either of p53, p63, or p73. Since NF-Y binds to many promoters besides the cdk1 and cyclin B promoters, inactivation of NF-Y by p53 family genes may be a general mechanism for transcription repression in replicative senescence.

Gewin L., Galloway D.A.

ABSTRACT Human papillomavirus type 16 (HPV-16) E6 activates telomerase specifically in epithelial cells. The oncogene c- myc has also been shown to activate telomerase in several cell types. Here we show that while both HPV-16 E6 and c- myc require intact E boxes to transactivate the hTERT promoter, E6 does not induce hTERT transcription simply by inducing expression of c- myc . Moreover, hTERT transactivation by HPV-16 E6 correlates with its ability to bind the cellular E6-associated protein (E6AP), suggesting that E6 and E6AP may target a regulator of hTERT expression.

Oh S.T., Kyo S., Laimins L.A.

ABSTRACT High-risk human papillomaviruses (HPVs) immortalize keratinocytes by disrupting the retinoblastoma protein (Rb)/p16 pathway and activating telomerase. The E7 oncoprotein targets Rb, while the E6 oncoprotein induces telomerase activity in human keratinocytes. This study has examined the mechanism by which E6 activates telomerase. Expression of human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, was found to be increased in keratinocytes stably expressing HPV type 16 E6, suggesting that E6 acts to increase hTERT transcription. hTERT expression and telomerase activity were activated to significantly higher levels in cells expressing both E6 and E7 than in cells expressing E6 alone. This indicates that E7 may augment E6-mediated activation of hTERT transcription. In transient-transfection assays using hTERT reporters, the induction of hTERT expression by E6 was found to be mediated by a 258-bp fragment of the hTERT promoter, proximal to the ATG initiation codon. Previous studies have demonstrated that overexpression of Myc can activate hTERT expression, suggesting that Myc may be a mediator of E6-mediated hTERT induction. However, in cells stably expressing E6, no strict correlation between the level of Myc and the activation of hTERT was found. Consistent with this observation, mutation of the two Myc binding sites in the hTERT promoter only modestly reduced responsiveness to E6 in transient reporter assays. This indicates that activation of Myc-dependent transcription is not essential for E6-mediated upregulation of hTERT expression. The hTERT promoter also contains five GC-rich elements that can bind Sp1. Mutation of these sites within the 258-bp fragment partially reduced hTERT induction by E6. However, when mutations in the Sp1 sites were combined with the mutated Myc binding sites, all activation by E6 was lost. This indicates that it is the combinatorial binding of factors to Myc and Sp1 cis elements that is responsible for hTERT induction by E6.

Itahana K., Dimri G., Campisi J.

Many normal cells respond to potentially oncogenic stimuli by undergoing cellular senescence, a state of irreversibly arrested proliferation and altered differentiated function. Cellular senescence very likely evolved to suppress tumorigenesis. In support of this idea, it is regulated by several tumor suppressor genes. At the heart of this regulation is p53. p53 is essential for the senescence response to short telomeres, DNA damage, oncogenes and supraphysiological mitogenic signals, and overexpression of certain tumor suppressor genes. Despite the well-documented central role for p53 in the senescence response, many questions remain regarding how p53 senses senescence-inducing stimuli and how it elicits the senescent phenotype.

Veldman T., Horikawa I., Barrett J.C., Schlegel R.

ABSTRACT The E6 and E7 oncogenes of human papillomavirus type 16 (HPV-16) are sufficient for the immortalization of human genital keratinocytes in vitro. The products of these viral genes associate with p53 and pRb tumor suppressor proteins, respectively, and interfere with their normal growth-regulatory functions. The HPV-16 E6 protein has also been shown to increase the telomerase enzyme activity in primary epithelial cells by an unknown mechanism. We report here that a study using reverse transcription-PCR and RNase protection assays in transduced primary human foreskin keratinocytes (HFKs) shows that the E6 gene (but not the E7 gene) increases telomerase hTERT gene transcription coordinately with E6-induced telomerase activity. In these same cells, the E6 gene induces a 6.5-fold increase in the activity of a 1,165-bp 5′ promoter/regulatory region of the hTERT gene, and this induction is attributable to a minimal 251-bp sequence (−211 to +40). Furthermore, there is a 35-bp region (+5 to +40) within this minimal E6-responsive promoter that is responsible for 60% of E6 activity. Although the minimal hTERT promoter contains Myc-responsive E-box elements and recent studies have suggested a role for Myc protein in hTERT transcriptional control, we found no alterations in the abundance of either c-Myc or c-Mad in E6-transduced HFKs, suggesting that there are other or additional transcription factors critical for regulating hTERT expression.

Campisi J.

Can studying cultured cells inform us about the biology of aging? The idea that this may be was stimulated by the first formal description of replicative senescence. Replicative senescence limits the proliferation of normal human cells in culture, causing them to irreversibly arrest growth and adopt striking changes in cell function. We now know that telomere shortening, which occurs in most somatic cells as a consequence of DNA replication, drives replicative senescence in human cells. However, rodent cells also undergo replicative senescence, despite very long telomeres, and DNA damage, the action of certain oncogenes and changes in chromatin induce a phenotype similar to that of replicatively senescent cells. Thus, replicative senescence is an example of the more general process of cellular senescence, indicating that the telomere hypothesis of aging is a misnomer, Cellular senescence appears to be a response to potentially oncogenic insults, including oxidative stress. The growth arrest almost certainly suppresses tumorigenesis, at least in young organisms, whereas the functional changes may contribute to aging, although this has yet to be critically tested. Thus, cellular senescence may be an example of antagonistic pleiotropy. Cross-species comparisons suggest there is a relationship between the senescence of cells in culture and organismal life span, but the relationship is neither quantitative nor direct.

Gong H., Wang P., Yu M., Zhu Y., Teng L., Su Y.

<b><i>Objectives:</i></b> Human papillomavirus 16 (HPV 16) E2 is a transcriptional regulator that plays a key role in regulating a variety of biological responses. Hematopoietic cell-specific protein 1-related protein X-1 (HAX-1) is a mitochondrial membrane protein, and the HAX-1 gene is involved in the occurrence, growth, invasion, and metastasis of various human malignant tumors. The purpose of this study was to investigate the relationships among HPV 16 E2, the role of HAX-1 gene, and the underlying intracellular apoptotic mechanism of human cervical squamous carcinoma cells (C33a and SiHa). <b><i>Methods:</i></b> In this study, HAX-1 expression was examined using real-time polymerase chain reaction, Western blot, and immunohistochemical staining analysis. Apoptosis of cells was assessed by flow cytometry. The mitochondrial function was assessed by the mitochondrial copy number, reactive oxygen species (ROS) generation, the mitochondrial membrane potential (ΔΨm), and mitochondrial morphology. <b><i>Results:</i></b> Our study demonstrated that the expression of the HAX-1 gene was significantly increased in human cervical carcinoma tissues relative to noncancerous cervix tissues. HPV 16 E2 inhibited HAX-1 protein expression. Overexpression of HAX-1 increased the mitochondrial copy number, decreased the production of ROS, and maintained the integrity of the mitochondrial membrane and morphology. So, enhanced expression of the HAX-1 gene could abrogate the HPV 16 E2-induced cell apoptosis. <b><i>Conclusion:</i></b> Therefore, these data support a mechanism that HAX-1 plays a crucial role in HPV 16 E2-induced human cervical squamous carcinoma cell apoptosis in a mitochondrial-dependent manner.

Gustinucci D., Ciccocioppo L., Coppola L., Negri G., Zannoni G., Passamonti B., Cesarini E., Ianzano C., Andreano T., Pireddu A., Giorgi-Rossi P.

Objective: To evaluate the clinical accuracy of Hepika test to identify cancer/precancerous lesions of the uterine cervix. Materials and Methods: A multicentre retrospective study was carried out in 2018 and included 330 liquid-based cytology samples from three Italian centres of women aged 25–64 who had been tested for the human papillomavirus (HPV) and whose histology or follow-up outcome was known. Hepika is an enzyme-linked immunosorbent assay (ELISA) targeting the protein complexes E6#p53 and E7#pRb. After excluding samples without sufficient residual material, the clinical accuracy of Hepika test was evaluated in 274 samples: adenocarcinoma (ADC) (4), squamous cell carcinoma (SCC) (7), adenocarcinoma in situ (AIS) (1), cervical intraepithelial neoplasia (CIN) grade 3 (60), CIN2 (51), CIN1 (34), and negative histology (117). Association, sensitivity, and specificity for carcinoma, CIN3+ and CIN2+ are reported. Results: Positive Hepika test was associated with a high probability of carcinoma (odds ratio (DOR) = 33.68, 95% confidence interval (CI) 7.0–163.1); sensitivity was 81.8%, specificity, 88.2%. A positive Hepika test showed a weaker association with CIN3+ lesions (DOR = 3.5; 95% CI 1.75–6.99) and lower sensitivity (27.8%). Conclusion: The Hepika test was found to be an accurate biomarker for HPV-induced cervical carcinoma. Population-based prospective studies are needed to confirm the clinical usefulness of the Hepika test in the differential diagnosis of HPV-induced invasive lesions.

Munk M., Alcalde J., Lorentzen L., Villalobo A., Berchtold M.W., Panina S.

Calmodulin (CaM) is the principle mediator of the Ca2+ signal in all eukaryotic cells. A huge variety of basic cellular processes including cell cycle control, proliferation, secretion and motility, among many others are governed by CaM, which regulates activities of myriads of target proteins. Mammalian CaM is encoded by three genes localized on different chromosomes all producing an identical protein. In this study, we have generated HeLa human cancer cells conditionally expressing CaM in a genetic background with all three genes inactivated by CRISPR/Cas9. We demonstrate that downregulation of ectopically expressed CaM is achieved after 120 h, when cells are arrested in the M phase of the cell cycle. We show for the first time that CaM downregulation in human cancer cells is followed by a multinucleated senescent state as indicated by expression of β-galactosidase as well as cell morphology typical for senescent cells. Our newly generated genetic system may be useful for the analysis of other CaM regulated processes in eukaryotic cells in the absence of endogenous CaM genes.

Domínguez-Catzín V., Reveles-Espinoza A., Sánchez-Ramos J., Cruz-Cadena R., Lemus-Hernández D., Garrido E.

Cervical cancer is the fourth cause of death worldwide by cancer in women and is a disease associated to persistent infection with human papillomavirus (HPV), particularly from two high-risk types HPV16 and 18. The virus initiates its replicative cycle infecting cells located in the basal layer of the epithelium, where a small population of epithelial stem cells is located performing important functions of renewal and maintenance of the tissue. Viral E2 gene is one of the first expressed after infection and plays relevant roles in the replicative cycle of the virus, modifying fundamental processes in the infected cells. Thus, the aim of the present study was to demonstrate the presence of hierarchic subpopulations in HaCaT cell line and evaluate the effect of HPV16-E2 expression, on their biological processes. HaCaT-HPV16-E2 cells were generated by transduction of HaCaT cell line with a lentiviral vector. The α6-integrin-CD71 expression profile was established by immunostaining and flow cytometric analysis. After sorting, cell subpopulations were analyzed in biological assays for self-renewal, clonogenicity and expression of stemness factors (RT-qPCR). We identified in HaCaT cell line three different subpopulations that correspond to early differentiated cells (α6-integrindim), transitory amplifying cells (α6-integrinbri/CD71bri) and progenitor cells (α6-integrinbri/CD71dim). The last subpopulation showed stem cell characteristics, such as self-renewal ability, clonogenicity and expression of the well-known stem cell factors SOX2, OCT4 and NANOG, suggesting they are stem-like cells. Interestingly, the expression of HPV16-E2 in HaCaT cells changed its α6-integrin-CD71 immunophenotype modifying the relative abundance of the cell subpopulations, reducing significantly the percentage of α6-integrinbri/CD71dim cells. Moreover, the expression of the stem cell markers was also modified, increasing the expression of SOX2 and NANOG, but decreasing notably the expression of OCT4. Our data demonstrated the presence of a small subpopulation with epithelial “progenitor cells” characteristics in the HaCaT cell line, and that HPV16-E2 expression on these cells induces early differentiation.

Provinciali M., Pierpaoli E., Piacenza F., Giacconi R., Costarelli L., Basso A., Recchioni R., Marcheselli F., Bray D., Benlhassan K., Malavolta M.

Cellular senescence is a complex response to stress that contributes to suppress cancer and to initialize mechanisms of repair after tissue injury. However, the accumulation of senescent cells is considered a hallmark of aging and is believed to contribute to the aging phenotype and to drive age-related pathologies. For these reasons, modulation of this response is emerging as a promising therapeutic target in cancer and aging research. In particular, it is desirable to induce cellular senescence in cancer cells for the purpose of an anticancer therapy but, conversely, it is likely that preventing excessive accumulation of senescent cells during aging could help for the purpose of health maintenance and longevity. In this chapter we review the evidence that cellular senescence can be effectively targeted in vitro by various dietary bioactive compounds and will attempt to elucidate the mechanisms involved. We also discuss the problems that hamper the translation of these findings into the development of nutraceuticals for therapeutic purposes in vivo.

Deftereos G., Kiviat N.B.

Papillomaviruses (PV) are extremely widespread, small, non-enveloped, icosahedral, species-specific, double-stranded DNA epitheliotropic viruses, which include human papillomaviruses (HPVs), known to infect humans. In humans, these viruses establish chronic epithelial infections, with each HPV type being associated with infection of specific anatomic sites and distinct natural histories. The clinical manifestations of HPV infection include genital and cutaneous warts as well as genital tract lesions referred to as “intraepithelial neoplasia.” Of greatest importance is that fact that a subset of HPVs (referred to as “high-risk” types of HPV) plays a central role in the development of most lower genital tract neoplasias as well as some head and neck carcinomas.

Khokhlov A.N.

Today, gerontologists usually employ certain molecular or cellular biomarkers of aging to evaluate the effects of various interventions in this process, since this approach is much more time-efficient than the construction of survival curves. However, arguments for the expediency of using such biomarkers are often based on the results of studies on what is called cell/cellular senescence. Unfortunately, the usage of this term has recently evolved so that it has largely lost its initial meaning, which is that normal cultured cells are subject to replicative senescence (according to the Hayflick phenomenon) and undergo changes similar to those in the cells of an aging organism. Most of recent studies in this field deal with the induction of relevant changes in cultured (usually transformed) cells by various DNA-damaging factors. Such an approach is important for defining the strategy of cancer control but, yet again, leads away from the study of actual mechanisms of organismal aging. Moreover, there are grounds to consider that biomarkers of aging identified in these studies (in particular, senescence-associated beta-galactosidase activity, the most popular among them) are basically linked to cell proliferative status. At the organismal level, this status is generally determined by the program of development and differentiation of tissues and organs, which in a definitive state are composed of postmitotic or very slowly propagating cells. Therefore, it appears that canceling the aging program will not cause any significant changes in the age-dependent dynamics of the above biomarkers. This conclusion brings us back to the necessity of constructing the survival curves for test groups of animals or humans as the only reliable (though expensive and time-inefficient) approach to evaluating the efficiency of means to modify the aging process.

Khokhlov A.N.

The term “cellular/cell senescence” was first introduced by Leonard Hayflick to describe the “age-related” changes in normal eukaryotic cells during aging in vitro, i.e., over the exhaustion of their mitotic potential. In the “classic” variant, it was assumed that cells “grow old” with the help of some internal mechanism, which leads to accumulation of various macromolecular defects (DNA damage in the first place). Currently, as a rule, “cellular senescence” means accumulation/appearance of particular “biomarkers of aging” in cells (they are most often transformed cells that do not demonstrate any replicative senescence) under the influence of various external factors (oxidative stress, H2O2, mitomycin C, ethanol, ionizing radiation, doxorubicin, etc.) that cause DNA damage. This phenomenon has been called DDR (DNA Damage Response). Among the said biomarkers, there are senescence-associated beta-galactosidase activity, expression of p53 and p21 proteins as well as of proteins involved in the regulation of inflammation, such as IL-6 or IL-8, activation of oncogenes, etc. Thus, “aging/senescence” of cells does not occur simply by itself—it takes place because of the influence of DNA-damaging agents. This approach, in my opinion, despite being very important to define a strategy to fight cancer, distracts us, yet again, from the study of the real mechanisms of aging. It should be emphasized that the “stationary phase aging” model developed in my laboratory also allows registering the occurrence of certain biomarkers of aging in cultured cells, but in this case they arise due to the restriction of their proliferation by contact inhibition, i.e., due to a rather physiological impact, which does not cause any damage to cells by itself (the situation is similar to what we observe in a whole multicellular organism).

Cho S., Hwang E.S.

SA β-Gal activity is a key marker of cellular senescence. The origin of this activity is the lysosomal β-galactosidase, whose activity has increased high enough to be detected at suboptimal pH. SA β-Gal is also expressed in the cells in quiescence driven by serum-starvation or a high confluency, and it has been hypothesized that SA β-Gal positivity is rather a surrogate marker of high lysosome content or activity. In this study, it was determined how SA β-Gal activity is expressed in quiescence and how lysosome content and activities are differently maintained in senescence and quiescence using DNA damage-induced senescence and serum starvation-induced quiescence as study models. Lysosome content increased to facilitate SA β-Gal expression in both the conditions but with a big difference in the levels of the change. Lipofuscins whose accumulation leads to an increase in residual bodies also increased but with a smaller difference between the two conditions. Meanwhile, lysosome biogenesis was actively ongoing only in senescence progression, indicating that the difference in the lysosome contents may largely be due to lysosome biogenesis. Further, the cells undergoing senescence progression but not the ones in quiescence maintained high mTOR and low autophagy activities. Overall, the results indicate that, although SA β-Gal is expressed due to the elevated lysosome content in both cellular senescence and quiescence, senescence differs from quiescence with high lysosome biogenesis and low autophagy activity, and mTOR activity might be involved in these differences.

Burns J.E., Walker H.F., Schmitz C., Maitland N.J.

Expression of the HPV E2 open reading frame in cervical cancer cells has been shown to affect the expression of both viral and cellular genes. We have examined the phenotypic effects of the expression of human papillomavirus 16 E2 open reading frame in the human keratinocyte cell line HaCaT. Increased levels of apoptotic cell death were seen within 24h of the transfection of HPV-16 E2 expression constructs. However, in those cells which survived selection and retained the intact E2 ORF, long-term stable expression of E2, as detected by RT-PCR, produced cells which developed phenotypes typical of terminally differentiated cells. These included characteristic morphological changes and expression of involucrin, filaggrin and senescence markers. This provides the first evidence of a role for E2 in stimulation of the normal epithelial differentiation programme, which would promote the progression of the HPV life cycle.

DiMaio D., Johung K., Goodwin E., Horner S., Yates K.

Mayeaux E.J., Dunton C.

External genital warts are a significant health problem particularly for young adults. This review summarizes the current literature on epidemiology, transmission, diagnosis, and treatment. Efficacy of all treatments is less than optimal, and multiple therapies may be necessary for complete resolution. Data on a new patient-applied therapy are presented. New vaccine therapy for prevention of infection should reduce the incidence of disease.

Münger K., Howley P., DiMaio D.

Johung K., Goodwin E.C., DiMaio D.

ABSTRACT This work demonstrates a central role for the retinoblastoma (Rb) family in driving the transcriptional program of induced and replicative senescence. HeLa cervical carcinoma cells rapidly undergo senescence when the human papillomavirus (HPV) type 18 E7 gene in these cells is repressed by the bovine papillomavirus (BPV) E2 protein. This senescence response requires the endogenous Rb pathway but not the p53 pathway. Microarray analysis 6 days after BPV E2 introduction into HeLa cells identified 224 cellular genes induced by E7 repression and 354 repressed genes. Many repressed genes were involved in cell cycle progression, and numerous induced genes encoded lysosomal proteins. These gene expression changes were blocked by constitutive expression of the wild-type HPV16 E7 or adenovirus E1A gene, but not by E7 or E1A mutants defective for Rb binding. Short hairpin RNAs targeting the Rb family also inhibited these gene expression changes and blocked senescence. Therefore, surprisingly, the transcriptional response to BPV E2 expression was entirely dependent on E7 repression and activation of the Rb family, and the BPV E2 protein did not directly affect the expression of cellular genes. Activation of the Rb family repressed E2F-responsive genes and stimulated transcriptional activators, thereby mobilizing multiple signals, such as repression of B-MYB and DEK, that were independently sufficient to induce senescence. There was extensive overlap between the transcriptional profiles of senescent, late-passage primary human fibroblasts and senescent cervical carcinoma cells, suggesting that this Rb family-mediated transcriptional cascade also plays a central role in replicative senescence.

Nishimura A., Nakahara T., Ueno T., Sasaki K., Yoshida S., Kyo S., Howley P.M., Sakai H.

Most human papillomavirus (HPV)-positive cervical cancers contain integrated copies of the viral genome in their chromosomes and express the viral oncoproteins E6 and E7. A virus-encoded transcription factor, E2, is known to repress E6/E7 expression in HPV-positive cancer cells, leading to growth inhibition, which indicates that E6/E7 is required for the survival of the cells. We found that the E2-mediated growth inhibition of HeLa cells, an HPV18-positive cancer cell line, was coupled with a reduction in telomerase activity, an effect which was rescued by the complementation of E7 expression, but not E6 expression, indicating that the cell viability and the telomerase activity in HeLa cells are maintained by an E7-associated function. Analysis of E7 mutants suggested that the binding to the pRB family of pocket proteins was involved in the ability of E7 to rescue the growth potential and telomerase activity inhibited by E2 expression. We also showed that the telomerase activity upregulated by E7 expression was determined by the hTERT promoter activity, and that c-Myc upregulation caused by pRB inactivation could account for the promoter activity. The activation of p53 and consequent accumulation of p21Cip1, which were triggered by the downregulation of E6, appeared not to be essential for the E2-mediated growth arrest.