Inhibition of cervical cancer cell growth in vitro and in vivo with lentiviral-vector delivered short hairpin RNA targeting human papillomavirus E6 and E7 oncogenes

Gu, Wenyi; Putral, Lisa N.; Hengst, Kylie; Minto, Kelly; Saunders, Nicholas A.; Leggatt, Graham R.; McMillan, Nigel A.J.

doi:10.1038/sj.cgt.7700971

Cited by 113 publications

(111 citation statements)

References 42 publications

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“…Low-dose E7-shRNA infection of HeLa cells with lentiviral vectorshRNA caused reduced cell growth and the induction of senescence, whereas a high-dose infection resulted in specific cell death via apoptosis. 33 Similar results also have been reported that the Sulindac, a nonsteroidal anti-inflammatory drug (NSAID), induces specific degradation of the HPV oncoprotein E7 and causes growth arrest and apoptosis in cervical carcinoma cells. 34 Even with all these information available, the oncogenic mechanism of E7 still remains elusive basically because the mutant forms of E7 that still maintain complete pRb-binding ability fail to transform cells.…”

Section: Discussionsupporting

confidence: 72%

Increased expression of Dyrk1a in HPV16 immortalized Keratinocytes enable evasion of apoptosis

Chang

Lin

Yang

et al. 2007

Intl Journal of Cancer

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Immortalization is a critical event in virus-related oncogenesis. No enough information, however, is currently available to elucidate the changes that occur in cellular molecules during immortalization. To identify potential cellular markers or regulators involving in immortalization, a paired-cell model of primary foreskin keratinocytes (FK) and HPV16 immortalized foreskin keratinocytes were established. Using mRNA differential display, RT-PCR and Northern blot methods, we have identified and confirmed that Dyrk1a (dual-specificity tyrosine-phosphorylated and regulated kinase 1A) is present and increased in HPV16 immortalized cells, but is absent in primary keratinocytes. Moreover, transfection of E7 siRNA oligo into immortalized cells leads to a diminishing E7 expression and the eventual disappearance of Dyrk1a. Similar results of Dyrk1a expressional differences could also be seen when tissue specimens were compared using LCM/real-time PCR and immunohistochemistry analysis; malignant cervical lesions contain significantly more DYRK1A than normal tissue. It was also demonstrated that raised DYRK1A could rearrange the cellular localization of FKHR (forkhead in rhabdomyosarcoma), an apoptosis activator, and suppress BAD. Importantly, this phenomenon can be reversed when endogenous Dyrk1a was knocked down in immortalized cells by RNA interference. These results suggest that the raised Dyrk1a in HPV16 immortalized keratinocytes and cervical lesions may serve as a candidate antiapoptotic factor in the FKHR regulated pathway and initiate immortalization and tumorigenesis gradually. ' 2007 Wiley-Liss, Inc.Key words: HPV16; keratinocytes; Dyrk1a; immortalization; apoptosis Mammalian cells have a limited life span and proliferating capacity when cultivated in vitro. Replicate senescence or the terminal arrest state, has been found to be accompanied by several molecular changes that lead to an activated suppression of the cell cycle and the shortening of the telomeres. 1 Similar to apoptosis, cellular senescence is thought to be a mechanism of tumor suppression because it prevents the outgrowth of cells that have acquired mutations in genes rendering them cancerous. Consistent with this model, several tumor suppressor genes (such as, overexpressed p16 or ZNF217, activated p53 and deregulated Rassf1a or c-myc, etc) or their products have been reported and shown to be associated in parallel with telomere irregularities at the onset of cellular senescence. 2 Telomere shortening has been shown to generate unstable DNA leading to p53 activation and the subsequent transcriptional induction of the cdk inhibitor p21 CIP13 that moves cells into senescence state. Therefore, preventing telomere erosion by ectopic expression of telomerase is sufficient to immortalized primary cells in vitro and thus extend life span. [4][5][6] Although it works in human fibroblast, telemorase alone, however, is not sufficient to induce immortalization in human keratinocytes and mammary epithelial cells. 7 To identify other factors that are involved ...

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Section: Discussionsupporting

confidence: 72%

Increased expression of Dyrk1a in HPV16 immortalized Keratinocytes enable evasion of apoptosis

Chang

Lin

Yang

et al. 2007

Intl Journal of Cancer

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“…Green fluorescent protein (GFP)-expressing FaDu cells (FaDu-GFP) were established following infection with the GFP-expressing pLL3.7 lentiviral construct as previously described (10,11). Cells were checked at intervals through fluorescence microscopy or fluorescence-activated cell sorting to confirm continued stable expression of GFP.…”

Section: Generation Of Green Fluorescent Protein-expressing Cells Andmentioning

confidence: 99%

Cisplatin Treatment Induces a Transient Increase in Tumorigenic Potential Associated with High Interleukin-6 Expression in Head and Neck Squamous Cell Carcinoma

Poth

Guminski

Thomas

et al. 2010

Molecular Cancer Therapeutics

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Head and neck squamous cell carcinoma (HNSCC) is characterized by the 5-year survival rate of ∼50%. Despite aggressive surgical, radiation, and chemotherapeutic interventions, 30% to 40% of patients die from the development of recurrent or disseminated disease that is resistant to chemotherapy. As a model of recurrence, we examined the effects of cisplatin on the ability of head and neck cancer cells to initiate tumors in a xenotransplant model. HNSCC cells were treated in vitro with cisplatin at a concentration that elicited >99% cytotoxicity and assessed for tumorigenic potential in nonobese diabetic/severe combined immunodeficient mice. HNSCC cells that survived cisplatin treatment formed tumors in nonobese diabetic/severe combined immunodeficient mice more efficiently than nontreated cells. Cisplatin-resistant cells were characterized using clonal analysis, in vivo imaging, and transcriptomic profiling. Preliminary functional assessment of a gene, interleukin-6 (IL-6), highly upregulated in cisplatin-treated cells was carried out using clonogenicity and tumorigenicity assays. We show that cisplatin-induced IL-6 expression can contribute to the increase in tumorigenic potential of head and neck cancer cells but does not contribute to cisplatin resistance. Finally, through clonal analysis, we show that cisplatin-induced IL-6 expression and cisplatin-induced tumorigenicity are stochastically derived. We report that cisplatin treatment of head and neck cancer cells results in a transient accumulation of cisplatin-resistant, small, and IL-6-positive cells that are highly tumorigenic. These data also suggest that therapies that reduce IL-6 action may reduce recurrence rates and/or increase disease-free survival times in head and neck cancer patients, and thus, IL-6 represents a promising new target in HNSCC treatment. Mol Cancer Ther; 9(8); 2430-9. ©2010 AACR.

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“…[8][9][10] Therefore, these two oncogenes are regarded as ideal targets for developing and testing RNAi-based therapeutic treatments. Previous studies, including our own, have demonstrated that silencing HPV E6/E7, with siRNA or short hairpin RNA (shRNA), leads to cervical cancer cells undergoing apoptosis or senescence [11][12][13][14] and inhibition of tumor growth in vivo. [14][15][16][17] These results suggest that RNAi therapy could be developed for cervical cancer treatment.…”

Section: Introductionmentioning

confidence: 99%

Inhibition of cervical cancer cell growth in vitro and in vivo with dual shRNAs

Payne

Sun

et al. 2010

Cancer Gene Ther

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RNA interference (RNAi)-based gene silencing is widely used in laboratories for gene function studies and also holds a great promise for developing treatments for diseases. However, in vivo delivery of RNAi therapy remains a key issue. Lentiviral vectors have been employed for stable gene transfer and gene therapy and therefore are expected to deliver a stable and durable RNAi therapy. But this does not seem to be true in some disease models. Here, we showed that lentivirus delivered short-hairpin RNA (shRNA) against human papillomavirus (HPV) E6/E7 oncogenes were effective for only 2 weeks in a cervical cancer model. However, using this vector to carry two copies of the same shRNA or two shRNAs targeting at two different but closely related genes (HPV E6 and vascular endothelial growth factor) was more effective at silencing the gene targets and inhibiting cell or even tumor growth than their single shRNA counterparts. The cancer cells treated with dual shRNA were also more sensitive to chemotherapeutic drugs than single shRNA-treated cells. These results suggest that a multi-shRNA strategy may be a more attractive approach for developing an RNAi therapy for this cancer.

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Inhibition of cervical cancer cell growth in vitro and in vivo with lentiviral-vector delivered short hairpin RNA targeting human papillomavirus E6 and E7 oncogenes

Cited by 113 publications

References 42 publications

Increased expression of Dyrk1a in HPV16 immortalized Keratinocytes enable evasion of apoptosis

Increased expression of Dyrk1a in HPV16 immortalized Keratinocytes enable evasion of apoptosis

Cisplatin Treatment Induces a Transient Increase in Tumorigenic Potential Associated with High Interleukin-6 Expression in Head and Neck Squamous Cell Carcinoma

Inhibition of cervical cancer cell growth in vitro and in vivo with dual shRNAs

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