Summary Replicative senescence limits the proliferation of somatic cells passaged in culture and may reflect cellular aging in vivo. The most widely used biomarker for senescent and aging cells is senescence‐associated β‐galactosidase (SA‐β‐gal), which is defined as β‐galactosidase activity detectable at pH 6.0 in senescent cells, but the origin of SA‐β‐gal and its cellular roles in senescence are not known. We demonstrate here that SA‐β‐gal activity is expressed from GLB1, the gene encoding lysosomal β‐D‐galactosidase, the activity of which is typically measured at acidic pH 4.5. Fibroblasts from patients with autosomal recessive GM1‐gangliosidosis, which have defective lysosomal β‐galactosidase, did not express SA‐β‐gal at late passages even though they underwent replicative senescence. In addition, late passage normal fibroblasts expressing small‐hairpin interfering RNA that depleted GLB1 mRNA underwent senescence but failed to express SA‐β‐gal. GLB1 mRNA depletion also prevented expression of SA‐β‐gal activity in HeLa cervical carcinoma cells induced to enter a senescent state by repression of their endogenous human papillomavirus E7 oncogene. SA‐β‐gal induction during senescence was due at least in part to increased expression of the lysosomal β‐galactosidase protein. These results also indicate that SA‐β‐gal is not required for senescence.

Most cervical carcinomas express high-risk human papillomaviruses (HPVs) E6 and E7 proteins, which neutralize cellular tumor suppressor function. To determine the consequences of removing the E6 and E7 proteins from cervical cancer cells, we infected HeLa cells, a cervical carcinoma cell line that contains HPV18 DNA, with a recombinant virus that expresses the bovine papillomavirus E2 protein. Expression of the E2 protein resulted in rapid repression of HPV E6 and E7 expression, followed Ϸ12 h later by profound inhibition of cellular DNA synthesis. Shortly after E6͞E7 repression, there was dramatic posttranscriptional induction of p53. Two p53-responsive genes, mdm2 and p21, were induced with slightly slower kinetics than p53 and appeared to be functional, as assessed by inhibition of cyclin-dependent kinase activity and p53 destabilization. There was also dramatic posttranscriptional induction of p105 Rb and p107 after E6͞E7 repression, followed shortly thereafter by induction of p130. By 24 h after infection, only hypophosphorylated p105 Rb was detectable and transcription of several Rb͞E2F-regulated genes was dramatically repressed. Constitutive expression of the HPV16 E6͞E7 genes alleviated E2-induced growth inhibition and impaired activation of the Rb pathway and repression of E2F-responsive genes. This dynamic response strongly suggests that the p53 and Rb tumor suppressor pathways are intact in HeLa cells and that repression of HPV E6 and E7 mobilizes these pathways in an orderly fashion to deliver growth inhibitory signals to the cells. Strikingly, the major alterations in the cell cycle machinery underlying cervical carcinogenesis can be reversed by repression of the endogenous HPV oncogenes. H igh-risk human papillomaviruses (HPVs) such as HPV18 play a central role in the development of essentially all cases of cervical carcinoma (1). However, carcinoma develops infrequently even after infection by these HPV types, and it typically occurs years to decades after the initial infection. Two HPV oncogenes, E6 and E7, are expressed in cervical carcinomas and carcinoma-derived cell lines. The E6 and E7 proteins can immortalize cultured primary human keratinocytes, but these immortalized cells are not tumorigenic unless additional, undefined genetic events occur. These observations imply that the viral oncogenes do not directly induce tumor formation but rather set in motion a series of events that may ultimately result in tumorigenicity.The high-risk HPV E6 and E7 proteins exert profound effects on the tumor suppressor proteins p53 and p105 Rb (1). These tumor suppressor proteins normally control signaling pathways that regulate the cell cycle and monitor and protect the integrity of the genome. p53 is a transcription factor that activates transcription of a variety of genes including p21Waf1/CIP1/SDI1 (p21) (reviewed in ref.2). p21 directly inhibits the activity of cyclin-dependent kinase (cdk) complexes, which are required for cell cycle progression. Transcription of the mdm2 gene is also induced by p53. m...

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.Cervical carcinoma is initiated by infection with a high-risk human papillomavirus (HPV), usually HPV type 16 (HPV16) or HPV18, and gene transfer studies have identified the E6 and E7 genes as the major viral oncogenes (62). The HPV E6 and the E7 proteins modulate cellular proteins that regulate the cell cycle (reviewed in references 35 and 38). The E6 protein binds to the p53 tumor suppressor protein and targets it for accelerated ubiquitin-mediated degradation. The E6 protein also stimulates telomerase activity in cultured keratinocytes. The E7 protein binds to the active, hypophosphorylated form of p105Rb and other members of the retinoblastoma (Rb) family of tumor suppressor proteins, resulting in their destabilization and loss of Rb/E2F complexes that repress transcription of genes required for cell cycle progression. The p53 and Rb pathways are interconnected: cyclin-dependent kinase (cdk)-mediated phosphorylation of p105 Rb , which also disrupts Rb/E2F complexes, is inhibited by p21, a transcriptional target of p53. The E6 and E7 proteins also appear to have p53-and Rb-independent activities (35,38).The high-risk HPV E6 and E7 proteins have profound effects on the growth properties of primary cultured human keratinocytes. Normal human keratinocytes undergo a limited number of divisions in culture before they enter replicative senescence, an irreversible, nonproliferative state characterized by growth factor-resistant growth arrest, specific morphological changes, increased autofluorescence, and elevated senescence-associated ␤-galactosidase (SA␤-Gal) activity (48). Escape from replicative senescence and immortalization of cultured human ker...

Trafficking of human papillomaviruses to the Golgi apparatus during virus entry requires retromer, an endosomal coat protein complex that mediates the vesicular transport of cellular transmembrane proteins from the endosome to the Golgi apparatus or the plasma membrane. Here we show that the HPV16 L2 minor capsid protein is a retromer cargo, even though L2 is not a transmembrane protein. We show that direct binding of retromer to a conserved sequence in the carboxy-terminus of L2 is required for exit of L2 from the early endosome and delivery to the trans-Golgi network during virus entry. This binding site is different from known retromer binding motifs and can be replaced by a sorting signal from a cellular retromer cargo. Thus, HPV16 is an unconventional particulate retromer cargo, and retromer binding initiates retrograde transport of viral components from the endosome to the trans-Golgi network during virus entry. We propose that the carboxy-terminal segment of L2 protein protrudes through the endosomal membrane and is accessed by retromer in the cytoplasm.

Expression of the bovine papillomavirus E2 regulatory protein in human cervical carcinoma cell lines repressed expression of the resident human papillomavirus E6 and E7 oncogenes and within a few days caused essentially all of the cells to synchronously display numerous phenotypic markers characteristic of cells undergoing replicative senescence. This process was accompanied by marked but in some cases transient alterations in the expression of cell cycle regulatory proteins and by decreased telomerase activity. We propose that the human papillomavirus E6 and E7 proteins actively prevent senescence from occurring in cervical carcinoma cells, and that once viral oncogene expression is extinguished, the senescence program is rapidly executed. Activation of endogenous senescence pathways in cancer cells may represent an alternative approach to treat human cancers. Cancer is thought to be caused by the stepwise accumulation of genetic changes, resulting in progressive phenotypic abnormality. Infection of cervical keratinocytes with a highrisk human papillomavirus (HPV) such as HPV18 is the initiating event in the great majority of cervical carcinomas (1). The HPV E6 and E7 proteins are continuously expressed in cervical carcinoma cells and seem to play a role in tumor development and in maintenance of the malignant phenotype (1). The role of the E6 and E7 proteins in the progression from normal keratinocytes to transformed cells has been studied in cell culture. Normal human keratinocytes can undergo a limited number of cell divisions in vitro and then stop proliferating, a phenomenon referred to as replicative senescence (2-4). Telomere length progressively shortens during cell passage (5, 6), and it has been proposed that once the length of one or more telomeres falls below a critical threshold, an active genetic program is mobilized that culminates in senescence (7,8). Cultured keratinocytes can escape senescence and become immortalized by inactivation of the retinoblastoma tumor suppressor pathway and activation of telomerase, the enzyme responsible for maintaining telomere length (9, 10). Expression of the HPV E6 and E7 proteins also can immortalize keratinocytes (11,12), an activity that is consistent with the known biochemical activities of these proteins. The E7 protein neutralizes the retinoblastoma tumor suppressor pathway, and the E6 protein stimulates telomerase activity in keratinocytes (1,6,13). Although the mechanistic basis for this effect on telomerase activity is not known, this activity seems crucial for immortalization because E6 mutants defective for telomerase activation are immortalization-defective (10). In addition, host-cell mutations are required for HPVimmortalized keratinocytes to become tumorigenic and to acquire other malignant characteristics, such as metastatic potential (9, 14 -20). The E6 and E7 proteins facilitate the acquisition of these mutations by causing accelerated degradation of p53 and p105 Rb , respectively, resulting in abrogation of DNA damage checkpoint control, elevat...