Cellular Mechanisms and Cancer BioFiles Issue 24.pdf

2007

VOLUME 2

NUMBER 4

FOR LIFE SCIENCE RESEARCH

INNOVATION @ WORK

ABC TRANSPORTERS AND

CANCER DRUG RESISTANCE

NF- k B AND

INFLAMMATION

DNA DAMAGE AND

REPAIR

NEW PRODUCTS

Cultured HeLa cells in metaphase stage

of mitosis.

Cellular Mechanisms

and Cancer

sigma.com

INNOVATION @ WORK

NEW from Sigma

Your comprehensive gene search tool

Your Favorite Gene ™ is a new search tool that matches your gene of

interest to thousands of research products available from Sigma.

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■ Use a wide variety of terms to locate genes

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■ 150,000 shRNAs

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■ 4,000 antibodies, proteins & kits

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■ Human Protein Reference Database

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Introduction

The medical definition of cancer appears simple and straightforward. According to the

U.S. National Cancer Institute, cancer is “(a) term for diseases in which abnormal cells

divide without control.” 1 Behind this basic definition is a complex and unpredictable

spectrum of over 100 types of cancer.

The human aspect of cancer cannot be completely separated from the scientific research.

The World Health Organization recognizes cancer as a leading cause of death worldwide,

and emphasizes prevention and early detection as crucial to reducing the global burden

of the disease. 2 The American Association for Cancer Research established its Scientist-

Survivor program in 1999 to encourage communication between patients, patient

advocates, and leading scientists in the field. 3 Newspapers, magazines, and other media

sources announce breakthrough discoveries to the public, increasing awareness that

cancer is not an individual disease but a collection that has no single initiating event or

defined evolution.

Cancer results from a cascade of abnormal cell reactions. When a cellular mechanism

goes wrong, the resulting damage, if not repaired, may contribute to a cell’s evolu-

tion into malignancy. Because cancers begin with a single cell, cancer investigators use

genomics, proteomics, and signaling techniques to determine and evaluate cellular

changes and contributing cause and effect. Discoveries such as the correlation between

the human papilloma virus and cervical cancer encourage the scientific community to

seek similar breakthroughs for other cancer types. The understanding of cellular muta-

tions and signaling pathways involved in mutagenesis and abnormal cell function has

been used to screen potential new drugs with more efficacy and/or less toxicity.

It’s impossible to comprehensively review current cancer research; the amount of informa-

tion is enormous and the rate of discovery is increasing. In the preface to his book “The

Biology of Cancer”, Robert Weinberg writes “…we are deluged with a vast amount of

genetic, biochemical, and cell biological information about cancer development, far more

almost than any human mind can assimilate and comprehend.” 4 For this issue of BioFiles

we have selected three aspects of cancer biology to review.

■ The exploitation of ABC transporter proteins by cancer cells to export

chemotherapeutic drugs

■ The activation of NF

FOR LIFE SCIENCE RESEARCH

2007

Volume 2

Number 4

Table of Contents

Cellular Mechanisms and Cancer

Innovation @ Work

MISSION ® shRNA Human Tumor

Suppressor Gene Panel ..................... 2

Panorama ® Antibody Arrays ..................... 3

GenomePlex ® Single Cell

Whole Genome Amplification ........... 4

GenomePlex ® Complete

Whole Genome Amplification ........... 5

PhosphoProfile™ I Phosphopeptide

Enrichment Kit .................................. 6

−κ

B in response to inflammation and its role in

ABC Transporters and Cancer Drug

Resistance

cancer progression

■ Genetic damage, mutagenesis, and cellular repair processes

Role of ABC Transporters and Multi-Drug

Resistance Reversal Using

RNA Interference...................................... 7

MDR-substrate Anticancer Agents............. 9

Innovation @ Work - Innovative products from Sigma for genomics and proteomics stud-

ies, including gene silencing, protein expression profiling, phosphopeptide enrichment,

and whole genome amplification, are also highlighted.

ABC Transporter Membrane Proteins...... 10

Antibodies to ABC Transporters............... 11

Compounds for MDR Detection .............. 13

References:

1. World Health Organization, Cancer Fact Sheet No. 297, Feb. 2006, www.who.int/mediacentre/

factsheets/fs297/en/index.html

2. National Cancer Institute, U.S. National Institutes of Health, www.cancer.gov

3. American Association for Cancer Research, www.aacr.org

4. The Biology of Cancer, Robert A. Weinberg, Garland Science, Taylor & Francis Group, LLC, New

York, NY (2007)

NF- k B and Inflammation

Inflammation and Cancer:

The NF- k B Connection ............................. 14

Natural Product Inhibitors of

NF- k B Activation ................................... 16

Antibodies and Kits ............................... 18

Cover image: Cultured HeLa cells labeled with anti-tubulin antibody and propidium iodide (to label

the DNA) in metaphase stage of mitosis. Photographed by Dr. K. G. Murti, St. Jude Children’s

Research Hospital, Memphis, TN.

Technical content: Libby Yunger, Ph. D., Chloe McClanahan, B. Sc., Vicki Caligur, B. Sc.

DNA Damage and Repair

DNA Damage and Repair Mechanisms.... 20

DNA Alkylating Agents ........................... 24

DNA Crosslinking Agents........................ 26

DNA Synthesis Inhibitors ........................ 27

DNA Repair Enzymes and Antibodies ...... 28

New Products

Antitumor Agents .................................. 30

Chemopreventative Agents ..................... 32

Enzymes................................................. 32

Multi-Drug Resistance ............................ 32

Tumor Growth Regulation....................... 32

For Safety Information please refer to the Sigma Biochemicals, Reagents and Kits for Life Science

Research Catalog or Sigma.com

INNOVATION @ WORK

MISSION ® shRNA Human Tumor Suppressor

Gene Panel

Silencing Tumor Suppressor Genes Using

MISSION ® TRC shRNA

RNAi methodologies have been utilized in small- to large-scale screening projects,

allowing researchers to perform gene-silencing experiments in timeframes and

target cells not previously possible. While siRNA screens have become fairly com-

mon, large-scale screening with shRNA is still evolving. Advantages of shRNA-based

experiments include long-term knockdown and viral delivery to nontransfectable

cell types.

The MISSION TRC shRNA Human Tumor Suppressor Gene Family Set is a panel of

lentiviral-based shRNAs consisting of approximately 75 gene sets, each represented

by 3–5 individual shRNA clones. The shRNA constructs included in the tumor sup-

pressor set allow you to identify genes involved in cell survival by assisting in mak-

ing a cell more sensitive, or more resistant, to an added chemotherapeutic agent

through the knockdown of gene expression and resulting function.

Scientists at Sigma have utilized the MISSION TRC shRNA Human Tumor Suppressor

Gene Family Set in order to address biologically relevant questions while simultane-

ously developing screening strategies that can be used by researchers in the field.

The tumor suppressor gene family set delivers a potential pharmacogenomics solu-

tion, allowing you to profile the gene expression of a cancer cell line and compare

it against the response of the cell line to chemotherapeutic treatment. 1

In our study of paclitaxel resistance by the human lung cancer cell line, A549,

various transcripts were down-regulated in the lung cancer cells using the tumor

suppressor set. Several genes that are known to correlate with paclitaxel resistance,

including p53 and VHL , were identified by the panel. In addition, some genes,

when down-regulated by these shRNAs, led to increased sensitivity of the cells to

paclitaxel treatment.

Rapidly validate and compare results

in multiple model systems – spend

more time analyzing your data and less

time gathering it

■ Simplified, ready to transduce lenti-

viral format – straightforward shRNA

protocols let you progress to new cell

line studies faster

■ shRNA enables you to down-regulate

genes long-term – easily transfer your

experiments from in vitro to in vivo

1. Ji, D., Deeds, S.L., and Weinstein, E.J., shRNAs

Targeting Tumor Suppressor Genes, Gen. Eng. News , 27 ,

26-27 (2007).

Ordering Information

Cat. No. Product Name

Pack Size

SH0531 MISSION TRC shRNA

Human Tumor Suppressors

1 set

SHP001 MISSION Lentiviral

Packaging Mix

0.25 mL

1.7 mL

H9268 Hexadimethrine bromide

5 g

10 g

50 g

100 g

Sense Strand

CCGG NNNNNNNNNNNNNNNNNNNNN CTCGAG NNNNNNNNNNNNNNNNNNNNN TTTTT

GGCC NNNNNNNNNNNNNNNNNNNNN GAGCTC NNNNNNNNNNNNNNNNNNNNN AAAAA

Antisense Strand

P9620

Puromycin Ready Made

Solution, 10 mg/mL in

water

10 mL

cppt

N4888 Nutrient Mixture

F-12 Ham with sodium

bicarbonate, without

L -glutamine, sterile-filtered

liquid, cell culture tested

500 mL

6 3 500 mL

24 3 500 mL

hPGK

RRE

puroR

QR0200 Quantitative RT-PCR

ReadyMix™

1 kit

pLKO.1-puro

7,091 bp

SIN/3’ LTR

1 mg

5 mg

25 mg

For more information, please visit our Web site at

sigma.com/rnai.

Paclitaxel from Taxus

brevifolia

(Ψ)

Psi

RSV/5' LTR

f1 ori

pUC ori

ampR

5' -

3' - UU

Sense Strand

Antisense Strand

Features of the pLKO.1-Puro vector allow for transient or stable transfection of the shRNA as

well as production of lentiviral particles. Unlike adenovirus or murine-based MML or MSCV ret-

roviral systems, lentiviral-based particles permit efficient infection and integration of the specific

shRNA construct into differentiated and nondividing cells, overcoming low transfection and

integration difficulties when using these cell lines. pLKO.1 allows for long-term knockdown

and phenotypic observation and transduction of difficult or sensitive cell lines.

sigma.com

Features and Beneﬁ ts

■

T7402

INNOVATION @ WORK

Panorama ® Antibody Arrays

How the Panorama Antibody Arrays Work

Protein Expression Proﬁ ling

Sample A

Sample B

Profile 224 protein targets in half a day

■ Antibodies react with human, mouse and rat species

■

Step1:

Extract proteins

Comprehensive kits include buffers, extraction reagents, analysis

software, and labware

Step 2:

Label samples with

Cy3/Cy5 and mix

High Throughput Protein Expression Analysis

Panorama Antibody Microarrays are designed to identify the changes in expres-

sion of multiple targets in a biological sample with a single assay. During cellular

processes, mRNA undergoes a number of alternative processing steps prior to

and following translation so there is often poor correlation between mRNA

expression profiles and protein expression. 1 Antibody arrays provide an effective

solution for analyzing biologically-relevant events at the proteome level.

Highly specific antibodies are arrayed in duplicate on nitrocellulose slides com-

patible with most DNA array scanners. Cy™3/Cy5-based detection chemistries

may be used with cell or tissue extracts. Array antibodies are available separately

for additional downstream analyses.

Step 3:

Incubate on the array

Step 4:

Scan the array

Induction with Retinoic Acid in Mouse F9 Cells

Applications

■ Sample response to external treatments (e.g., starvation, addition of growth

factors, serum, drugs/biomolecules)

■ Comparison of different cell or tissue samples (clinical specimens, tissue,

heart, brain, lung, etc.)

Non-Treated (Cy3)

Treated (Cy5)

■

Determine off-target effects in gene silencing (RNAi) studies

Up-Regulated

Down-Regulated

Components

Panorama Antibody Slides

Connexin 43

PAR4

–

QuadriPERM ® Cell Culture Vessel

Extraction/Labeling Buffer

Protease Inhibitor Cocktail

Polyglutamylated

Tubulin

Phosphatase Inhibitor Cocktail II

Benzonase ® Ultrapure

Grb2

–

Array Incubation Buffer

Collection Tubes, Polypropylene

Phosphate Buffered Saline, pH 7.4, with

TWEEN ® 20 (Washing Buffer)

SigmaSpin ™ Post-Reaction Clean-Up

Columns

β -Tubulin I

–

β -Tubulin IV

Panorama Antibody List on diskette

including analysis software

1. Gygi, S.P., et al., Mol. Cell Bio. , 19 , 1720-1730 (1999)

Unchanged

Fluorescence Intensity

Low level

High level

Using the Panorama Antibody Microarray – Cell Signaling

Kit, proteins from mouse F9 cells are observed to be up-

regulated or down-regulated in expected fashion following

stimulation with retinoic acid. F9 cells were treated for

96 hours with all-trans-retinoic acid (10 -7 M). Extracts were

prepared from untreated and treated cells using Extraction/

Labeling Buffer and labeled with Cy3 and Cy5, respectively.

A mixture containing equal amounts of each labeled extract

(5 µg/mL) was incubated on the array as described in the kit

protocol. The same slide is shown at the two fluorescence

emission wavelengths for Cy3 and Cy5. (Note that the blue

background for Cy5 represents the normal and unavoidable

interference of the nitrocellulose membrane.) Changes

in expression level were confirmed by immunoblot, as

indicated.

Ordering Information

Cat. No. Product Name

Pack Size

CSAA1

Panorama Antibody Microarray - Cell Signaling

1 kit

GRAA2

Panorama Antibody Microarray - Gene Regulations

1 kit

MPAA3

Panorama Antibody Microarray - MAPK & PKC

Pathways

1 kit

PPAA4

Panorama Antibody Microarray - p53 Pathways

1 kit

XP725

Panorama Antibody Microarray - XPRESS Profiler725

1 kit

For more information, please visit our Web site a t sigma.com/arrays.

Features

■ Rapid, multiplex analysis of protein expression

■

–

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