20 maart 2012: onderstaande laboratorium studies uit 2002 hebben veel vervolg gekregen in de afgelopen jaren. Onder dit artikel heb ik twee studieabstracten uit 2010 toegevoegd die bewijzen dat met name de blokkade van de eiwitten E 6 en E 7 effectief kan zijn in vormen van kanker veroorzaakt door het HPV virus. Echter het onderzoek blijft nog steeds beperkt tot laobartoriumonderzoek. Zie ook referentielijst onderaan.

30 november 2004: Bron: Yahoonews

Een eenvoudig aan te brengen gel werkt als gentherapie tegen baarmoederhalskankercellen. Engelse onderzoekers stopten met de gel de eiwitten E 6 en E 7 en daarmee werd 100 % massale apoptosis - zelfmoord van baarmoederhalskankercellen veroorzaakt. Interessant is dat deze studie een vervolg is op een eerdere studie uit 2002 en nu weer tot betere resultaten komt. Dit samengevoegd met het nieuws over het vaccin tegen HPV, zie ook onder artiklen hier op deze pagina, lijkt nu niet alleen aan middel als preventie binnen enkele jaren beschikbaar te komen maar ook een eenvoudig middel om baarmoederhalskanker tot stilstand te brengen en wellicht zelfs te genezen. Al is dit laatste na slechts enkele laboratoriumstudies natuurlijk nog prematuur.
Achtereenvolgens plaatsen we hier twee artikelen over zelfde studie gepubliceerd door Yahoo nieuws en een Engelstalige Medische site.

Gel may help cure cervical cancer
By Michael Le Page

It might be possible to combat early-stage cervical cancer simply by applying a gel to a woman’s cervix, rather than removing abnormal cells surgically. The team developing the gel hopes to carry out clinical trials in the UK soon, and similar gels could one day be developed to treat other cancers on accessible surfaces.
Cervical cancer kills almost a quarter of a million women worldwide each year. It is caused by a common sexually transmitted virus, the human papilloma virus (HPV).

Occasionally, the viral DNA inserts itself into the genome of cells on the surface of the cervix, boosting production of two viral proteins, E6 and E7. These prevent cell suicide and can turn cells cancerous. Several research groups have shown that if production of these proteins is blocked, cervical cancer cells growing in a dish commit suicide, while normal cells are unharmed. One way to block the proteins is to use RNA interference, which exploits an ancient natural defence mechanism against viruses.
If “short interfering RNAs” (siRNAs) – small pieces of RNA matching part of a specific gene – are introduced into cells, all RNA copies of that gene will be blocked before any protein can be made. Direct application

The big problem is delivery, because siRNAs are destroyed within minutes of injection into the bloodstream ( New Scientist print edition, 13 November, 2004). But Jo Milner at the University of York in the UK realised that because cervical cancers develop on or near the cervix’s surface, there might be no need to inject siRNAs.

Instead, her team is developing a gel that can be applied to the cervix directly. The gel contains siRNAs encased in liposomes, fatty particles that help them enter cells. Now Milner’s team has shown that the gel triggers apoptosis in cancerous cervical cells in culture. It is not clear whether the liposomes would penetrate deep enough into the cervix to kill off early-stage cancers. “I don’t want to raise false hopes,” says Milner. But she adds that the gel might work even if the siRNAs do not get into every cell, by tipping the balance in favour of the immune system.

In the long term, vaccines against HPV should slash the incidence of cervical cancer ( New Scientist print edition, 20 November 2004). But the new gel could prevent cells from turning cancerous in women who already have persistent infections, as well as killing tumours. Because there are far fewer safety concerns with locally applied therapies than with drugs that are swallowed or injected, Milner hopes UK regulators will soon give the go-ahead for tests on women with precancerous cells or early-stage cancers, before the routine surgical removal of abnormal cells.

Journal reference: Oligonucleotides (quarterly)

Hier een ander artikel over zelfde studie:

Bron: British Medical Journals

UK researchers switch off genes that trigger cervical cancer
Susan Mayor, London

UK researchers have reported that they have been able to switch off a human papillomavirus gene that triggers cervical cancer, using a new highly targeted techniqueRNA interference. A team at the University of York reported last week that RNA interference, which works by selectively "silencing" homologous genes, completely eliminated all human cervical cancer cells in vitro yet left healthy cells unharmed (Oncogene 2002;21: 6041-8).

This was the first evidence that RNA interference can turn off genes of infectious viruses in tumour cells, rendering them harmless. The researchers chose human cervical cancer cells that are positive for human papillomavirus type 16 for the experimental model because the type is well understood and clinically important. Over 90% of human cervical cancers test positive for papillomavirus, and abnormal cell proliferation is driven by the effects of two viral genes E6 and E7.

Professor Jo Milner, professor of cell biology at the University of York, and coworker Dr Ming Jiang tested whether RNA interference could silence E6 and E7 gene expression and restore normal cell functions. They designed short interfering RNA (siRNA) for each gene and used liposomes to introduce it into the cells.

The siRNA targeted homologous messenger RNA (mRNA)the intermediate messenger molecule that carries the code from DNA in cell nuclei to build proteins in the cell cytoplasmresulting in its destruction. RNA interference degrades mRNA and so blocks the production of the protein it codes for.
Professor Milner reported: "Silencing E6 completely eliminated E6 mRNA in cervical cancer cells, but the cells continued to grow, although at a slower rate. We then thought perhaps we might need to silence both E6 and E7."

Her research team tested siRNA for E7 alone to ensure that it worked. "To our surprise, silencing E7 completely stopped cell proliferation and caused massive apoptosis of the cells," she said. Apoptosis is the process of cell suicide that enables controlled removal of cells without inducing an inflammatory response. It is disrupted in many cancers, resulting in uncontrolled cell growth.

Reassuringly, the study showed that siRNA for E7 had no effect on healthy cells, showing its highly selective anticancer effect. "For the first time, we have demonstrated that siRNA can induce selective silencing of exogenous oncogenic viral genes in mammalian cells," Professor Milner explained. "Secondly, we have shown that the process of RNA interference does not interfere with the recovery of cellular regulatory systems previously inhibited by viral gene expression."

She added: "The research indicated that E7 siRNA has major therapeutic potential for the treatment, and possibly the prevention, of human cervical cancer."
The next step is to identify how E7 disrupts the normal apoptotic pathway. Many cancers involve an imbalance between processes that promote or inhibit cell death, so the proof that RNA interference can silence the genes leading to this loss of control could have wide applications.

Hier de eerdere studie uit 2002 gehaald uit Pubmed, die gedaan is met zelfde techniek door prof. dr. Jo Milner:

Selective silencing of viral gene expression in HPV-positive human cervical carcinoma cells treated with siRNA, a primer of RNA interference

Ming Jiang and Jo Milner
YCR P53 Research Group, Department of Biology, University of York, York YO10 5DD, UK Correspondence to: J Milner, E-mail: ajm24@york.ac.uk

Abstract
Selective silencing of mammalian gene expression has recently been achieved using short interfering RNA (siRNA). Synthetic siRNA targets homologous mRNA for degradation and the process is highly efficient. Here we demonstrate siRNA silencing of pathogenic viral gene expression. As a well characterized model we chose cervical carcinoma cells positive for human papillomavirus type 16. Over 90% of human cervical cancers are positive for papillomavirus and abnormal cell proliferation is driven by co-operative effects of viral E6 and E7 genes. We sought to silence HPV E6 and E7 gene expression using siRNAs to target the respective viral mRNAs. Our results indicate selective degradation of E6 and E7 mRNAs. Silencing was sustained for at least 4 days following a single dose of siRNA. E6 silencing induced accumulation of cellular p53 protein, transactivation of the cell cycle control p21 gene and reduced cell growth. In contrast, E7 silencing induced apoptotic cell death. HPV-negative cells appeared unaffected by the anti-viral siRNAs. Thus we demonstrate for the first time (i) that siRNA can induce selective silencing of exogenous viral genes in mammalian cells, and (ii) that the process of siRNA interference does not interfere with the recovery of cellular regulatory systems previously inhibited by viral gene expression.
Oncogene (2002) 21, 6041-6048. doi:10.1038/sj.onc.1205878

Repression of E6 and E7 oncogenes results in restoration of p53 and pRb suppressor pathways and induced apoptosis in HPV16-positive oropharyngeal squamous cell cancer cell lines

Bron: J Natl Cancer Inst.  2009 Mar 18;101(6):412-23. Epub 2009 Mar 10 Klik hier voor gratis inzage in het volledige studierapport.

E6 and e7 gene silencing and transformed phenotype of human papillomavirus 16-positive oropharyngeal cancer cells.

Source

Department of Medical Oncology, Yale University School of Medicine, New Haven, CT 06520, USA.

Abstract

BACKGROUND:

The E6 and E7 genes of human papillomavirus type 16 (HPV16) encode oncoproteins that bind and degrade p53 and retinoblastoma (pRb) tumor suppressors, respectively. We examined the effects of repressing E6 and E7 oncogene expression on the transformed phenotype of HPV16-positive oropharyngeal cancer cell lines.

METHODS:

Human oropharyngeal squamous cell cancer 147T and 090 (harboring integrated HPV16 DNA) and 040T (HPV DNA-negative) cells were infected with retroviruses that expressed a short hairpin RNA (shRNA) targeting the HPV16 E6 and E7 genes or a scrambled-sequence control shRNA. Flow cytometry, terminal deoxynucleotidyltransferase-mediated UTP end-labeling assay, and immunoblotting for annexin V were used to assess apoptosis in shRNA-infected cell lines. Biochemical analysis involved quantitative real-time polymerase chain reaction analysis of p53- and pRb-target gene expression and immunoblotting for p53 and pRb protein expression.

RESULTS:

In 147T and 090 cells, shRNA-mediated inhibition of HPV16 E6 and E7 expression reduced the E6 and E7 mRNA levels by more than 85% compared with control cells that expressed a scrambled-sequence shRNA. E6 and E7 repression resulted in restoration of p53 and pRB protein expression, increased expression of p53-target genes (p21 and FAS), decreased expression of genes whose expression is increased in the absence of functional pRb (DEK and B-MYB), and induced substantial apoptosis in 147T and 090 cells compared with the control shRNA-infected cells (from 13.4% in uninfected to 84.3% in infected 147T cells and from 3.3% in uninfected to 71.2% in infected 090 cells).

CONCLUSION:

Repression of E6 and E7 oncogenes results in restoration of p53 and pRb suppressor pathways and induced apoptosis in HPV16-positive oropharyngeal squamous cell cancer cell lines.

PMID:
19276448
[PubMed - indexed for MEDLINE]


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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.

Apoptosis. 2008 Feb;13(2):273-81.

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.

Source

Cancer Biology Research Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Ave., Wuhan, Hubei 430030, PR China.

Abstract

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.

PMID:
18060502
[PubMed - indexed for MEDLINE]


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