Characteristics of head and neck squamous cell carcinoma cell Lines reflect human tumor biology independent of primary etiologies and HPV status

Explanations for the differences in clinical outcomes in head and neck squamous cell carcinomas (HNSCCs) when compared by similar tumor location, stage, nodal status, human papillomavirus (HPV) status, and patient history remain elusive. Cell lines are an excellent tool of study for understanding the in vitro properties of cancers. However, HNSCC cell lines from progression-free and/or HPV-positive tumors are very rare. Here we studied HPV-positive and HPV-negative University of Michigan squamous cell carcinoma cell lines (2 HPV−, 2 HPV16+, 1 HPV18+) coming from donors with nonoropharyngeal sites and variant clinical outcomes. Cell morphology and proliferation were assessed, and immunofluorescence and Western blotting evaluated tumor biomarkers (TP53, RB1, p16, HPV E6 and E7, EGFR, Cyclin D1, Ki-67, and beta-catenin). Slow in vitro proliferation, long lag phase before exponential proliferation, lower maximal cell density, and higher wild-type TP53 expression were common to cell lines from patients who experienced long-term disease-free survival. In contrast, shorter lag phases, rapid proliferation, and high maximal cell density were observed in cell lines from patients who experienced aggressive tumor progression leading to death. Membrane-bound beta-catenin was present in all cell lines, but nuclear beta-catenin was associated with the more lethal cancers. In summary, the HNSCC cell lines present key characteristics, independent of primary etiologies and HPV infection, that mirror the behavior of the tumors from which they were derived.     

Detection of p53 protein and Ki-67 proliferation antigen in human papillomavirus (HPV)-positive and HPV-negative cervical lesions by immunohistochemical double-staining

In HPV-associated genital lesions, low or absent expression of p53 has been attributed to the rapid degradation of p53 through its binding with HPV E6 protein. In this study, we examined p53 protein expression with two antibodies (CM1 polyclonal and PAb 1801 monoclonal antibodies), and Ki-67 proliferation antigen (monoclonal antibody) using an immunohistochemical (IHC) double-staining technique in 77 HPV-positive cervical lesions (HPV6, HPV11, HPV16, HPV18, HPV31, and HPV33) and in 15 HPV-negative cases. p53 protein expression was detected in 36/92 (39.1%) of the specimens. of the p53-positive cases, 80.6% (29/36) were HPV-positive samples, including 10/23 (43.5%) of HPV16- and 3/10 (30%) of HPV18-positive biopsies. In 52.8% of the p53-positive samples, the expression was found in less than 5% of the basal cells which were also positive for Ki-67.
Ki-67 proliferation marker was found in 91/92 specimens, most intensely in those infected by HPV16. p53 was more abundant in progressive or persistent lesions, but no differences were found between HPV-positive and HPV-negative samples. the positive IHC double-staining of both p53 and Ki-67 proliferation antigen in the same basal (and parabasal) cells indicates that these two normal cell-cycle proteins are being expressed while the cells are entering from the G₁ to the S phase of the cell cycle. Since the latter property is only attributed to the wild-type p53 (but not to mutated p53), the p53 protein detected in HPV lesions by IHC is likely to be the wild-type p53 rather than mutated p53, and the result was also confirmed by using p53 mutant specific antibody PAb 240. Accordingly, the concept of HPV inactivating the wild-type p53 protein should be re-examined, and other mechanisms for HPV-mediated carcinogenesis should be considered.     

Down-regulation of p21 contributes to apoptosis induced by HPV E6 in human mammary epithelial cells

Infection with human papillomaviruses (HPV) is strongly associated with the development of cervical cancer. The HPV E6 gene is essential for the oncogenic potential of HPV. E6 induces cell proliferation and apoptosis in cervical cancer precursor lesions and in cultured cells. Although induction of telomerase and inactivation of the tumor suppressor p53 play important roles for E6 to promote cell growth, the molecular basis of E6-induced apoptosis is poorly understood. While it is expected that inactivation of p53 by E6 should lead to a reduction in cellular apoptosis, numerous studies demonstrated that E6 could in fact sensitize cells to apoptosis. Understanding the mechanism of p53-independent apoptosis is of clinical significance. In the present study, we investigated the mechanism of apoptosis during E6-mediated immortalization of primary human mammary epithelial cell (HMEC). E6 by itself is sufficient to immortalize HMECs and is believed to do so at least in part by activation of telomerase. During the process of E6-mediated HMEC immortalization, an increased apoptosis was observed. Mutational analysis demonstrated that E6-induced apoptosis was distinct from its ability to promote cell proliferation, activate telomerase, or degrade p53. While the known pro-apoptotic E6 target proteins such as Bak or c-Myc did not appear to play an important role, down-regulation of the cyclin-dependent kinase inhibitor p21^Waf1/Cip1 (p21) by E6 correlated with its ability to induce apoptosis. Ectopic expression of p21 inhibited E6-induced apoptosis. Moreover, a p53 degradation defective E6 mutant was competent for p21 down-regulation and apoptosis induction. The anti-apoptotic function of p21 may not simply be the result of p21-induced growth arrest. These studies demonstrate an E6 activity to down-regulate p21 that is important for induction of apoptosis.     

Effects of Iejimalide B, a marine macrolide, on growth and apoptosis in prostate cancer cell lines

Iejimalide B, a marine macrolide, causes growth inhibition in a variety of cancer cell lines at nanomolar concentrations. We have investigated the effects of Iejimalide B on cell cycle kinetics and apoptosis in the p53+/AR+ LNCaP and p53-/AR- PC-3 prostate cancer cell lines. Iejimalide B, has a dose and time dependent effect on cell number (as measured by crystal violet assay) in both cell lines. In LNCaP cells Iejimalide B induces a dose dependent G0/G1 arrest and apoptosis at 48 h (as measured by Apo-BrdU staining). In contrast, Iejimalide B initially induces G0/G1 arrest followed by S phase arrest but does not induce apoptosis in PC-3 cells. qPCR and Western analysis suggests that Iejimalide B modulates the steady state level of many gene products associated with cell cycle (including cyclins D, E, and B and p21(waf1/cip1)) and cell death (including survivin, p21B and BNIP3L) in LNCaP cells. In PC-3 cells Iejimalide B induces the expression of p21(waf1/cip1), down regulates the expression of cyclin A, and does not modulate the expression of the genes associated with cell death. Comparison of the effects of Iejimalide B on the two cell lines suggests that Iejimalide B induces cell cycle arrest by two different mechanisms and that the induction of apoptosis in LNCaP cells is p53-dependent.     

Therapeutic strategies of different HPV status in Head and Neck Squamous Cell Carcinoma

Head and neck squamous cell carcinoma (HNSCC) is the 9^th most common malignant tumor in the world. Based on the etiology, HNSCC has two main subtypes: human papillomavirus (HPV) -related and HPV-unrelated. HPV-positive HNSCC is more sensitive to treatment with favorable survival. Due to the different biological behaviors, individual therapy is necessary and urgently required to deduce the therapeutic intensity of HPV-positive disease and look for a more effective and toxicity-acceptable regimen for HPV-negative disease. EGFR amplification and PI3K/AKT/mTOR pathway aberrant activation are quite common in HPV-positive HNSCC. Besides, HPV infection alters immune cell infiltrating in HNSCC and encompasses a diverse and heterogeneous landscape with more immune infiltration. On the other hand, the chance of HPV-negative cancers harboring mutation on the P53 gene is significantly higher than that of HPV-positive disease. This review focuses on the updated preclinical and clinical data of HPV-positive and HPV-negative HNSCC and discusses the therapeutic strategies of different HPV status in HNSCC.     

Alpha-tocopheryl succinate induces cytostasis and apoptosis in osteosarcoma cells: the role of E2F1

Alpha-tocopheryl succinate (alpha-TOS), a redox-inactive analog of vitamin E, induces cell cycle arrest, differentiation, and triggers apoptosis. We examined the ability of alpha-TOS to induce cytostasis and/or apoptosis in two human osteosarcoma cell lines, which carry wild-type pRb but differ in the p53 status. In the wt-p53 cells, alpha-TOS induced apoptosis, which was associated with p53 activation and enhanced E2F1 expression. Mutant p53 cells failed to undergo apoptosis when challenged with alpha-TOS. The cell growth arrest after alpha-TOS treatment was associated with a reduced expression of E2F1. Knocking down E2F1 rendered the alpha-TOS-sensitive cells rather resistant to the apoptotic stimulus inducing a marked and prolonged cell growth arrest. We conclude that alpha-TOS induces cell growth arrest or apoptosis involving E2F1.     

JNK supports survival in melanoma cells by controlling cell cycle arrest and apoptosis

JNK1/2 proteins belong to the family of stress-activated protein kinases. They play a complex role in growth regulation, inducing either cell death or growth support. In this report, we provide evidence that, in human melanoma cells, JNK inhibition with the small molecule inhibitor SP600125 induces either predominantly a G2/M arrest or apoptosis depending on the cell line. In 1205Lu cells, JNK inhibition induced cell cycle arrest through p53-dependent induction of p21 Cip1/Waf1 expression, while in WM983B cells, induction of apoptosis by JNK inhibition was accompanied by p53, Bad and Bax induction, not p21 Cip1/Waf1. JNK inhibition with the small molecule inhibitor SP600125 slowed growth of all cell lines, although the effect was markedly greater in cells exhibiting high phospho- (P-)JNK1 levels. Specific gene knockdown of JNK1 by means of siRNA oligonucleotides inhibited cell growth only in melanoma cell lines exhibiting high P-JNK1 levels. siRNAs directed against JNK2 did not reduce cell growth in any of the cell lines tested. Together, our findings demonstrate that JNK, and in particular the JNK1 isoform, support the growth of melanoma cells, by controlling either cell cycle progression or apoptosis depending on the cellular context.     

Different cell cycle mechanisms between UV-induced and X-ray-induced apoptosis in WiDr colorectal carcinoma cells

Although DNA-damaging agents such as ultraviolet (UV) and X-ray can induce apoptosis, the difference in the apoptotic mechanism is not clearly understood. In the present study, we investigated the effects of these two genotoxic agents on the induction of DNA damage and subsequent apoptotic cell death from the viewpoint of cell cycle regulation by using WiDr cells. Transient G1 arrest was observed after UV exposure, whereas G2 but not G1 arrest was induced after X-ray irradiation. UV-exposure could induce G1 arrest in both mutant-type (mt-p53) and wild-type p53 (wt-p53) cells, but obvious G1 arrest was not observed in the cells lacking in p53 expression. An increase in the DNA fragmentation was observed at S phase in UV-irradiated cells and at G2 phase in X-irradiated cells, respectively. UV-irradiated cells showed an increase production of p53 protein and accumulation of p21 protein. On the contrary, both p53 and p21 proteins remained at a low level in X-irradiated cells. Treatment with aphidicolin, an S phase blocking agent, prolonged cell cycle arrest and reduced the rate of apoptotic cell death in both UV-irradiated and X-irradiated cells. From these results, it is suggested that UV-induced apoptosis occurs mainly at S phase and is regulated by increased production of p53 and p21 proteins, while X-ray-induced apoptosis occurs after G2 blockade and may be independent of p53.     

Human papillomavirus (HPV) 16 E6 sensitizes cells to atractyloside-induced apoptosis: Role of p53, ICE-like proteases and the mitochondrial permeability transition

Infection of cervical epithelial cells with certain high risk HPV genotypes is thought to play an etiologic role in the development of cervical cancer. In particular, HPV type 16 and 18 early protein 6 (E6) is thought to contribute to epithelial transformation by binding to the tumor suppressor protein p53, targeting it for rapid proteolysis, resulting in loss of its cell cycle arrest and apoptosis-inducing activities. Recent data indicate that factors responsible for triggering apoptosis reside in the cytoplasm of cells, and not in the nucleus. In particular, the findings that mitochondria are required in certain cell-free models for induction of apoptosis and that bcl-2 is localized to mitochondria have focused attention on the role of the mitochondrial membrane permeability transition (MPT) in apoptosis. Here we present data to indicate that HPV 16 E6 expression sensitizes cells to MPT-induced apoptosis. We also report that HPV 16 E6 sensitization of cells to MPT-induced apoptosis occurs only in the presence of wildtype (wt) p53 expression. The extent of apoptosis induced by atractyloside (an inducer of the MPT) in normal, temperature-sensitive (ts) p53, and HPV-16 E6 transfected J2-3T3 cells, and the HPV expressing cervical carcinoma cell lines SiHa, Hela and CaSki was determined. C33A cells, which express mutant p53 but not HPV, were also exposed to atractyloside in the presence or absence of HPV 16 E6 expression. Dose-dependent apoptosis induced by atractyloside in normal J2-3T3 cells and cervical carcinoma cells was measured by loss of cell viability, nuclear fragmentation and DNA laddering. The sensitivity of cells to atractyloside-induced apoptosis was found to be: HPV 16 E6-J2-3T3 > CaSki > normal-J2-3T3 cells ≈ ts p53-J2-3T3 ≈ vector-J2-3T3 cells > Hela > SiHa > C33A ≈ C33A 16 E6. Cyclosporin A (CsA), an inhibitor of the MPT, and ICE-I, a protease inhibitor, provided protection against atractyloside-induced apoptosis. These findings indicate that: 1) high risk HPV 16 E6 protein is capable of sensitizing cells to apoptosis; 2) HPV 16 E6 sensitization of cells to atractyloside-induced apoptosis occurs in a p53-dependent fashion; 3) the target of HPV 16 E6 sensitization of cells to atractyloside-induced apoptosis is the mitochondria; and 4) HPV 16 E6 sensitization of cells to atroctycoside-induced apoptosis involves an ICE-like protease-sensitive mechanism, regulating the onset of the MPT. These findings constitute the first evidence that mitochondria play a role in HPV 16 E6 modulation of apoptosis. J. Cell. Biochem. 66:245-255. © 1997 Wiley-Liss, Inc.     

Association of the ERK1/2 and p38 kinase pathways with nitric oxide-induced apoptosis and cell cycle arrest in colon cancer cells

To investigate the mechanism by which nitric oxide (NO) induces cell death in colon cancer cells, we compared two types of colon cancer cells with different p53 status: HCT116 (p53 wild-type) cells and SW620 (p53-deficient) cells. We found that S-nitrosoglutathione (GSNO), the NO donor, induced apoptosis in both types of colon cancer cells. However, SW620 cells were much more susceptible than HCT116 cells to apoptotic death by NO. We investigated the role of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 kinase on NO-induced apoptosis in both types of colon cancer cells. GSNO treatment effectively stimulated activation of the ERK1/2 and p38 kinase in both types of cells. In HCT116 cells, pretreatment with PD98059, an inhibitor of ERK1/2, or SB203580, an inhibitor of p38 kinase, had no marked effect on GSNO-induced apoptosis. However, in SW620 cells, SB203580 significantly reduced the NO-induced apoptosis, whereas PD098059 increases NO-induced apoptosis. Furthermore, we found evidence of cell cycle arrest of the G₀/G₁ phase in SW620 cells but not in HCT116 cells. Inhibition of ERK1/2 with PD098059, or of p38 kinase with SB203580, reduced the GSNO-induced cell cycle arrest of the G₀/G₁ phase in SW620 cells. We therefore conclude that NO-induced apoptosis in colon cancer cells is mediated by a p53-independent mechanism and that the pathways of ERK1/2 and p38 kinase are important in NO-induced apoptosis and in the cell cycle arrest of the G₀/G₁ phase.     

p53 arrests growth and induces differentiation of v-Myb-transformed monoblasts

Abstract The p53 protein can control cell cycle progression, programmed cell death, and differentiation of many cell types. Ectopic expression of p53 can resume capability of cell cycle arrest, differentiation, and apoptosis in various leukemic cell lines. In this work, we expressed human p53 protein in v-Myb-transformed chicken monoblasts. We found that even this protein possessing only 53% amino acid homology to its avian counterpart can significantly alter morphology and physiology of these cells causing the G2-phase cell cycle arrest and early monocytic differentiation. Our results document that the species-specific differences of the p53 molecules, promoters/enhancers, and co-factors in avian and human cells do not interfere with differentiation- and cell cycle arrest promoting capabilites of the p53 tumor suppressor even in the presence of functional v-Myb oncoprotein. The p53-induced differentiation and cell cycle arrest of v-Myb-transformed monoblasts are not associated with apoptosis suggesting that the p53-driven pathways controlling apoptosis and differentiation/proliferation are independent.     

Cisplatin restores p53 function and enhances the radiosensitivity in HPV16 E6 containing SiHa cells

Most HPV-positive cervical cancer cells possess wild type p53 gene, but its normal p53 functions are disrupted by expression of HPVs E6. Treatment with 0-20 microM cisplatin for 24 h in HPV16 E6 containing SiHa cells suppressed E6 mRNA, reduced E6 protein, and restored p53 expression in dose-dependent manners. Dual-parameter flow cytometric analysis indicated that sub-G(1) apoptotic cells, but not necrotic cells were the major species for cisplatin-induced cytotoxicity in SiHa cells. After 0-10 microM cisplatin treatment, slightly more apoptotic cells appeared from SiHa cells than those from dominant negative p53-transfected SiHa cells. There was no different ionizing radiation (IR)-induced apoptosis in these two different cells. On the other hand, cisplatin enhanced more IR-induced sub-G(1) apoptosis in SiHa than mp53-SiHa cells. These accompanied with prolonged p53 restoration in irradiated-SiHa cells after 24 h cisplatin treatment and thereafter. In contrast, it was not found in cells after irradiation alone. Similar results were also shown in Mdm2 expression in SiHa cells after combined treatment. Therefore, cisplatin restored p53 expression and prolonged IR-induced p53 restoration would be possible candidates to response more sub-G(1) apoptosis in irradiated SiHa cells. These results provided another new explanation on cisplatin sensitizing radiotherapy for HPV16 E6 containing cancer cells.     

Bortezomib sensitises TRAIL-resistant HPV-positive head and neck cancer cells to TRAIL through a caspase-dependent,E6-independent mechanism

Human papillomavirus (HPV) is causative for a new and increasing form of head and neck squamous cell carcinomas (HNSCCs). Although localised HPV-positive cancers have a favourable response to radio-chemotherapy (RT/CT), the impact of HPV in advanced or metastatic HNSCC remains to be defined and targeted therapeutics need to be tested for cancers resistant to RT/CT. To this end, we investigated the sensitivity of HPV-positive and -negative HNSCC cell lines to TRAIL (tumour necrosis factor-related apoptosis-inducing ligand), which induces tumour cell-specific apoptosis in various cancer types. A clear correlation was observed between HPV positivity and resistance to TRAIL compared with HPV-negative head and neck cancer cell lines. All TRAIL-resistant HPV-positive cell lines tested were sensitised to TRAIL-induced cell death by treatment with bortezomib, a clinically approved proteasome inhibitor. Bortezomib-mediated sensitisation to TRAIL was associated with enhanced activation of caspase-8, -9 and -3, elevated membrane expression levels of TRAIL-R2, cytochrome c release and G2/M arrest. Knockdown of caspase-8 significantly blocked cell death induced by the combination therapy, whereas the BH3-only protein Bid was not required for induction of apoptosis. XIAP depletion increased the sensitivity of both HPV-positive and -negative cells to TRAIL alone or in combination with bortezomib. In contrast, restoration of p53 following E6 knockdown in HPV-positive cells had no effect on their sensitivity to either single or combination therapy, suggesting a p53-independent pathway for the observed response. In summary, bortezomib-mediated proteasome inhibition sensitises previously resistant HPV-positive HNSCC cells to TRAIL-induced cell death through a mechanism involving both the extrinsic and intrinsic pathways of apoptosis. The cooperative effect of these two targeted anticancer agents therefore represents a promising treatment strategy for RT/CT-resistant HPV-associated head and neck cancers.Head and neck squamous cell carcinoma (HNSCC) represents the sixth most common cancer worldwide.¹ While the overall incidence of HNSCC, traditionally associated with tobacco or alcohol consumption, is declining, a subset of oropharyngeal cancers caused by infection with high-risk types of human papillomavirus (HPV) has risen significantly.^2,3 Transformation upon HPV infection occurs mainly because of inactivation of the p53 and retinoblastoma tumour suppressor proteins mediated by the viral oncoproteins E6 and E7, respectively.⁴HPV-positive (HPV⁺) cancers represent a distinct subset of HNSCC in terms of biology and clinical behaviour. In general, they are characterised by better overall survival and an improved response to conventional radio-chemotherapy (RT/CT) compared with HPV-negative (HPV⁻) cancers.^5,6 To further minimise treatment-related toxicity without compromising outcome, there have been suggestions of treatment de-escalation in conjunction with targeted therapies.⁷The novel anticancer agent TRAIL (tumour necrosis factor-related apoptosis-inducing ligand) selectively kills several types of malignant cell lines with little effect on normal cells.⁸ Recombinant TRAIL or monoclonal antibodies targeting TRAIL receptors (TRAIL-Rs) are currently being tested in phase I/II clinical trials for patients with advanced tumours.^9,10 TRAIL induces cell death by binding to TRAIL-R1 or TRAIL-R2, resulting in receptor oligomerisation and formation of the death-inducing signalling complex (DISC)¹¹ and activation of initiator caspase-8.¹² Caspase-8 directly activates effector caspase-3 to induce apoptosis through the type I pathway or cleaves the BH3-only protein Bid, generating tBid. This type II pathway involves an amplification loop through the intrinsic pathway of apoptosis characterised by cytochrome c release from the mitochondria, activation of initiator caspase-9 and ultimately caspase-3.¹³Despite its tumour-selective activity, various cancer cell lines remain resistant to TRAIL, limiting the clinical potential of TRAIL-based monotherapies. Many recent studies focus on combination strategies with other agents to sensitise resistant cells to TRAIL.¹⁴ The proteasome inhibitor bortezomib is an FDA-approved drug for the treatment of multiple myeloma, but has shown only little single-agent activity in solid malignancies such as HNSCC while being effective in combination with other treatment options.^{15, 16, 17} Combining bortezomib with TRAIL-R agonists produced a synergistic cytotoxic effect in various types of cancers. Potential mechanisms underlying sensitisation to TRAIL-induced apoptosis include inhibition of NF-κB signalling, stabilisation of BH3-only proteins, p53 or p21, upregulation of TRAIL-Rs and enhanced stability of caspase-8.^{18, 19, 20, 21, 22, 23, 24, 25, 26}So far, little data is available on the therapeutic potential of TRAIL alone or in combination with bortezomib in HNSCC or other HPV⁺ related cancers. Treatment with the proteasome inhibitor MG132 sensitised TRAIL-resistant HPV⁺ cervical cancer cells to TRAIL through p53-dependent upregulation of TRAIL-Rs and inactivation of XIAP.²⁷ Overexpression of E6 was shown to protect colon cancer cells from death receptor-induced apoptosis by affecting the stability of the DISC, indicating a functional link between the presence of E6 and TRAIL signalling.²⁸In this study, we tested the response of HPV⁺ and HPV⁻ HNSCC cells to treatment with TRAIL alone or combined with bortezomib, revealing a clear pattern of sensitivity to TRAIL depending on HPV status and a synergistic effect when combined with bortezomib. In addition, we identified some of the proteins and pathways involved in the response to TRAIL/bortezomib in HNSCCs.     

The human papillomavirus (HPV) 16 E2 protein induces apoptosis in the absence of other HPV proteins and via a p53-dependent pathway

The human papillomavirus (HPV) E2 protein regulates viral gene expression and is also required for viral replication. HPV-transformed cells often contain chromosomally integrated copies of the HPV genome in which the viral E2 gene is disrupted. We have shown previously that re-expression of the HPV 16 E2 protein in HPV 16-transformed cells results in cell death via apoptosis. Here we show that the HPV 16 E2 protein can induce apoptosis in both HPV-transformed and non-HPV-transformed cell lines. E2-induced apoptosis is abrogated by a trans-dominant negative mutant of p53 or by overexpression of the HPV 16 E6 protein, but is increased by overexpression of wild-type p53. We show that mutations that block the DNA binding activity of E2 do not impair the ability of this protein to induce apoptosis. In contrast, removal of both N-terminal domains from the E2 dimer completely blocks E2-induced cell death. Heterodimers formed between wild-type E2 and N-terminally deleted E2 proteins also fail to induce cell death. Our data suggest that neither the DNA binding activity of E2 nor other HPV proteins are required for the induction of apoptosis by E2 and that E2-induced cell death occurs via a p53-dependent pathway.     

RNA interference against HPV16 E7 oncogene leads to viral E6 and E7 suppression in cervical cancer cells and apoptosis via upregulation of Rb and p53

The simultaneous expression of human papillomavirus type 16 (HPV16) E6 and E7 oncogenes is pivotal for malignant transformation and maintenance of malignant phenotypes. Silencing these oncogenes is considered to be applicable in molecular therapies of human cervical cancer. However, it remains to be determined whether HPV16 E6 and E7 could be both silenced to obtain most efficient antitumor activity by using RNA interference (RNAi) technology. Herein, we designed a small interfering RNA (siRNA) targeting HPV16-E7 region to degrade either E6, or truncated E6 (E6*) and E7 mRNAs and to simultaneously knockdown both E6 and E7 expression. Firstly, the sequence targeting HPV16-E7 region was inserted into the shRNA packing vector pSIREN-DNR, yielding pSIREN-16E7 to stably express corresponding shRNA. HPV16-transformed SiHa and CaSki cells were used as a model system; RT-PCR, Western Blotting, MTT assay, TUNEL staining, Annexin V apoptosis assay and flow cytometry were applied to examine the effects of pSIREN-16E7. Our results indicated that HPV16-E7 specific shRNA (16E7-shRNA) induced selective degradation of E6 and E7 mRNAs and proteins. E6 silencing induced accumulation of cellular p53 and p21. In contrast, E7 silencing induced hypophosphorylation of retinoblastoma (Rb) protein. The loss of E6 and E7 reduced cell growth and ultimately resulted in massive apoptotic cell death selectively in HPV-positive cancer cells, compared with the HPV-negative ones. We demonstrated that 16E7-shRNA can induce simultaneous E6 and E7 suppression and lead to striking apoptosis in HPV16-related cancer cells by activating cellular p53, p21 and Rb. Therefore, RNAi using E7 shRNA may have the gene-specific therapy potential for HPV16-related cancers.     

p53 gene status modulates the chemosensitivity of non-small cell lung cancer cells

This study examined the effects of p53 gene status on DNA damage-induced cell death and chemosensitivity to various chemotherapeutic agents in non-small cell lung cancer (NSCLC) cells. A mutant p53 gene was introduced into cells carrying the wild-type p53 gene and also vice versa to introduce the wild-type p53 gene into cells carrying the mutant p53 gene. Chemosensitivity and DNA damage-induced apoptosis in these cells were then examined. This study included five cell lines, NCI-H1437, NCI-H727, NCI-H441 and NCI-H1299 which carry a mutant p53 gene and NCI-H460 which carries a wild-type p53 gene. Mutant p53-carrying cells were transfected with the wild-type p53 gene, while mutant p53 genes were introduced into NCI-H460 cells. These p53 genes were individually mutated at amino acid residues 143, 175, 248 and 273. The representative cell line NCI-H1437 cells transfected with wild-type p53 gene (H1437/wtp53) showed a dramatic increase in susceptibility to three anticancer agents (7-fold to cisplatin, 21-fold to etoposide, and 20-fold to camptothecin) compared to untransfected or neotransfected H1437 cells. An increase in chemosensitivity was also observed in wild-type p53 transfectants of H727, H441, H1299 cells. The results of chemosensitivity were consistent with the observations on apoptotic cell death. H1437/wtp53 cells, but not H1437 parental cells, exhibited a characteristic feature of apoptotic cell death that generated oligonucleosomal-sized DNA fragments. In contrast, loss of chemosensitivity and lack of p53-mediated DNA degradation in response to anticancer agents were observed in H460 cells transfected with mutant p53. These observations suggest that the increase in chemosensitivity was attributable to wild-type p53 mediation of the process of apoptosis. In addition, our results also suggest that p53 gene status modulates the extent of chemosensitivity and the induction of apoptosis by different anticancer agents in NSCLC cells.     

The roles of E6-AP and MDM2 in p53 regulation in human papillomavirus-positive cervical cancer cells

The p53 tumor suppressor is regulated by the MDM2 oncoprotein through a negative feedback mechanism. MDM2 promotes the ubiquitination and proteasome-dependent degradation of p53, possibly by acting as a ubiquitin ligase. In cervical cancer cells containing high-risk human papillomaviruses (HPV), p53 is also targeted for degradation by the HPV E6 oncoprotein in combination with the cellular E6-AP ubiquitin ligase. In this report, we describe the identification of efficient antisense oligonucleotides against human E6-AP. The roles of MDM2 and E6-AP in p53 regulation were investigated using a novel E6-AP antisense oligonucleotide and a previously characterized MDM2 antisense oligonucleotide. In HPV16-positive and HPV-18 positive cervical cancer cells, inhibition of E6-AP, but not MDM2, expression results in significant induction of p53. In HPV-negative tumor cells, p53 is activated by inhibition of MDM2 but not E6-AP. Furthermore, treatment with both E6-AP and MDM2 antisense oligonucleotides in HPV-positive cells does not lead to further induction of p53 over inhibition of E6-AP alone. Therefore, E6-AP-mediated degradation is dominant over MDM2 in cervical cancer cells but does not have a significant role in HPV-negative cells.     

p53-mediated cell death: relationship to cell cycle control.

M1 clone S6 myeloid leukemic cells do not express detectable p53 protein. When stably transfected with a temperature-sensitive mutant of p53, these cells undergo rapid cell death upon induction of wild-type (wt) p53 activity at the permissive temperature. This process has features of apoptosis. In a number of other cell systems, wt p53 activation has been shown to induce a growth arrest. Yet, wt 53 fails to induce a measurable growth arrest in M1 cells, and cell cycle progression proceeds while viability is being lost. There exists, however, a relationship between the cell cycle and p53-mediated death, and cells in G1 appear to be preferentially susceptible to the death-inducing activity of wt p53. In addition, p53-mediated M1 cell death can be inhibited by interleukin-6. The effect of the cytokine is specific to p53-mediated death, since apoptosis elicited by serum deprivation is refractory to interleukin-6. Our data imply that p53-mediated cell death is not dependent on the induction of a growth arrest but rather may result from mutually incompatible growth-regulatory signals.