An Recombinant Adenoviral System For Tetracycline-Regulated Reversible Cell Cycle Arrest, 12/02/2001

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Information about An Recombinant Adenoviral System For Tetracycline-Regulated Reversible...

Published on September 3, 2008

Author: organprinter

Source: slideshare.net

Description

An recombinant adenoviral system for
tetracycline-regulated reversible
cell cycle arrest
Wettergreen MA, Hunniford JW, Crawford JM, Adami GR.
The ability to regulate the proliferation of normal cells in a reversible manner would be a
useful adjunct to some clinical therapies, including many types of cancer chemotherapy.
While the application of recombinant growth factors and cytokines to target cells is a logical
approach to regulate cell proliferation, the high turnover rates of these peptide factors often
make this approach impractical. Recombinant adenoviral vectors can be used to direct the
expression of transgene products such as growth factors and cell cycle inhibitors in many
cell types in vitro and in vivo. We first used the tetracycline-regulated expression system to
allow regulated p16INK4a expression using recombinant adenovirus. We found that cell
cycle arrest was inefficient and to a large degree irreversible likely due to toxic effects of the
recombinant virus vector. This suggested a recombinant virus that produced a extracellular
acting factor would be more efficacious. We generated a tetracycline regulated recombinant
adenovirus capable of producing TGF-ß1 in cells. We demonstrate that infection with a
recombinant TGF-ß1-encoding virus system in primary human oral keratinocytes and in a
lung epithelial cell line is sufficient to allow a cell cycle arrest that is reversible upon
tetracycline addition. This inhibition is efficient even after the infection of a minority of cells in a
population. These results highlight the possibility of using low level infection with
recombinant adenovirus to cause short-term blocks on cell proliferation.

An Adenoviral System for Tetracycline-Regulated TGF- β Expression Mediates a Reversible Cell Cycle Arrest Wettergreen MA, Adami GR Department of Oral Medicine and Diagnostic Sciences, College of Dentistry, University of Illinois at Chicago, Chicago IL 60607. gadami@uic.edu Cell cycle specific chemotherapeutic drugs damage highly proliferative non- cancerous cells Rapidly proliferating epithelial cells of the oral mucosa develop oral mucositis within 4-7 days after chemotherapy treatment - Characterized by reddening, ulceration, infection, sloughing of cells Addition of TGF- β has been shown to protect oral mucosa against some chemotherapeutic drugs by arresting proliferation in G1 Problems with regulation of delivery of TGF-β preclude its usage in prevention of oral mucositis Adenoviral vectors have been used to deliver regulated amounts of growth factors to infected areas Regulated expression of these vectors has been accomplished with tetracycline system Here we detail creation f an adenoviral system used to deliver TGF- β to infected area regulated by addition of tetracycline - Dual virus system and single virus constructed Adenovirus Construction Replication-deficient E1- and E3-negative adenovirus created using standard recombination techniques AdtTA: expresses tetracycline-regulated transactivator tTA taken from pRETROOFF inserted in cloning site of pGEMCMV NEW AdTetTGF-B: expresses tetracycline regulated promoter (pRETROOFF) driving transcription of TGF-B cDNA Cysteine 223 and 225 changed to serines to make TGF-B into active form instead of latent Virus cesium banded and titered by plaque assay Dual virus system showed stable, reversible arrest of CCL-64 and oral keratinocytes Proliferation of cells returned to normal levels 1-2 days following tetracycline addition after TGF- β Infection of 10% total cells with both viruses and 30% infection with one of viruses resulted in sufficient TGF-B to block proliferation Low level of infection will reduce toxicity Levels of proliferation indicate protection from oral mucosa would be obtained - Inhibition of proliferation seen with as little as 50ng/ml TGF- β though more is being synthesized Single virus extremely unstable possibly due to large expression cassette Repression of proliferation with single virus may have contributed to poor amplification of single virus The authors acknowledge, Dr. JR Nevins for the gift of pGEMCMV NEW, and Dr. F. Graham for the gift of AdHCMVap1LacZ. Special thanks to Dr. JM Crawford for the oral keratinocytes and John Hunniford for help with the work Routine isolates of single virus after recombination lost activity with plaque purification Control at same titer had no effect and it was blocked by tetracyline. Figure 7. Growth inhibition of CCL-64 cells after infection with increasing MOI’s with single virus system. MOIs of 30 and 10 and 3 did nothing. Figure 3. Infection efficiency in CCL-64. Mock infection (A) or infection (B) at a MOI of 3.8 with AdHCMVap1LacZ that encodes β-galactosidase enzyme. A colorimetric assay for β-galactosidase activity 24 hours post-infection was performed and the cells were viewed with phase-contrast microscopy A B Figure 6. Tetracycline dependent reversibility of TGF- β production in normal human primary oral keratinocytes infected with AdtTA and AdTetTGF- β . Infection at MOI of 3/3. 48 h post-infection one set of cells was exposed to 10ug/ml tetracycline. Media was collected from both experiments and mean levels of active TGF- β were quantitated by ELISA. ELISA was performed twice in duplicate. DAY 0 DAY 2 DAY 5 -TET DAY 5 +TET Figure 4. Tetracycline-dependent reversibility of cell cycle inhibition in primary oral keratinocytes infected with AdtTA and AdTetTGF- β . 3 HT incorporated at 44 h post-infection. Percentage of cells entering S-Phase was quantified using autoradiography. Black nuclei indicate S-phase entry during each labeling period. A parallel plate was treated with/out tetracycline 48 h post-infection and then with and without tetracycline. Bar = 80 μm. Background and Significance Figure 2. Tetracycline-dependent reversibility of cell cycle inhibition in CCL-64 cells by AdtTA and AdTetTGF-B. Cells infected at MOI 1.8/2.0. Addition of tetracycline occurred at 2 day post-infection. Values shown are based on 4-10 data points. Results – Dual virus system Methods Acknowledgements Cellular Assays CCL-64 cells infected with dual virus system and exposed to 3H-T at 2,3,4,5,6 day post infection At 2 days post infection, tetracycline added to half of wells Cells fixed and evaluated with autoradiography to determine percentage of S- Phase entry Normal human oral keratinocytes infected at MOI of 3/3 with dual virus system 1-3 days after plating Cells exposed to 3H-T at 2,3,4,5 days post-infection Cells fixed and evaluated with HT3 and autoradiography for percentage of S- Phase entry A B Conclusions + 100 - 30 - 10 - 3 50% inhibition MOI ITR POLY A TGF- β cDNA P TRE ADENOVIRUS ITR ITR POLY A tTA cDNA P CMV ADENOVIRUS ITR Figure 1. Structure of recombinant adenovirus expressing TGF-B with TET-Off system Figure 5. Quantification of colorimetry experiment of normal primary keratinocytes. Values are means S.D. of 4 trials and are normalized to cells mock infected or infected with AdtTA virus alone. Results – Single virus

Cell cycle specific chemotherapeutic drugs damage highly proliferative non-

cancerous cells

Rapidly proliferating epithelial cells of the oral mucosa develop oral

mucositis within 4-7 days after chemotherapy treatment

- Characterized by reddening, ulceration, infection, sloughing of cells

Addition of TGF- β has been shown to protect oral mucosa against some

chemotherapeutic drugs by arresting proliferation in G1

Problems with regulation of delivery of TGF-β preclude its usage in

prevention of oral mucositis

Adenoviral vectors have been used to deliver regulated amounts of growth

factors to infected areas

Regulated expression of these vectors has been accomplished with

tetracycline system

Here we detail creation f an adenoviral system used to deliver TGF- β to

infected area regulated by addition of tetracycline

- Dual virus system and single virus constructed

Adenovirus Construction

Replication-deficient E1- and E3-negative adenovirus created using standard

recombination techniques

AdtTA: expresses tetracycline-regulated transactivator tTA taken from

pRETROOFF inserted in cloning site of pGEMCMV NEW

AdTetTGF-B: expresses tetracycline regulated promoter (pRETROOFF)

driving transcription of TGF-B cDNA

Cysteine 223 and 225 changed to serines to make TGF-B into active form

instead of latent

Virus cesium banded and titered by plaque assay

Dual virus system showed stable, reversible arrest

of CCL-64 and oral keratinocytes

Proliferation of cells returned to normal levels 1-2

days following tetracycline addition after TGF- β

Infection of 10% total cells with both viruses and

30% infection with one of viruses resulted in

sufficient TGF-B to block proliferation

Low level of infection will reduce toxicity

Levels of proliferation indicate protection from oral

mucosa would be obtained

- Inhibition of proliferation seen with as little as

50ng/ml TGF- β though more is being synthesized

Single virus extremely unstable possibly due to

large expression cassette

Repression of proliferation with single virus may

have contributed to poor amplification of single

virus

Routine isolates of single virus after

recombination lost activity with plaque

purification

Control at same titer had no effect and it was

blocked by tetracyline.

Cellular Assays

CCL-64 cells infected with dual virus system and exposed to 3H-T at 2,3,4,5,6 day post infection

At 2 days post infection, tetracycline added to half of wells

Cells fixed and evaluated with autoradiography to determine percentage of S-

Phase entry

Normal human oral keratinocytes infected at MOI of 3/3 with dual

virus system 1-3 days after plating

Cells exposed to 3H-T at 2,3,4,5 days post-infection

Cells fixed and evaluated with HT3 and autoradiography for percentage of S-

Phase entry

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