Cigarette Smoke Affects Your Genes and Then You

Francis AkkarapattiakalHuman Biology, Faculty of Arts & Science and

Medicine, University of Toronto

(Inhibition of PLCB2 Gene Increases Risk of Cancer)

Abstract• The principle objective of the study was to study the effects

of Cigarette Smoke Condensate (CSC )on human lung cells and then using microarray analysis to explore the genes and study their properties. The genes obtained were then analysed via MAGIC Tool Software to determine the top 20 up-regulated genes and top 20 down-regulated genes . The results showed a wide range of genes with various cellular/molecular functions present. The genes were also found to be at various locations when compared with other microarray analysis data. Of all the genes, PLCB2 was selected and studied to see the effect CSC has upon it. It was hypothesized that CSC down-regulates the gene and thus disrupts cellular motility and cell cycle progression.

Introduction - CSC

• Cigarette smoking is a major risk factor for numerous diseases, including cancer and diseases of the cardiovascular and respiratory systems1

• Major components of CSC are: Alkaloids, Nitrosamines, Polyaromatic Hydro Carbons

https://smokingtpckproject.files.wordpress.com/2012/09/smoking-ingredients.jpg

Introduction – Human Cancer Cell Lines• H1299 is a non small cell lung cancer line• Human Lung Epithelial Cell line used to evaluate effects of CSC on lung epithelial cell junction levels 2

• The results of this study demonstrated that CSC induced the loss of cellular junctions in lung epithelium 2

• Possibly a consequence of Connexin-4HNE formation- Connexin a protein used in intracellular communication

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4354730/figure/fig1/

Introduction – Lab Purpose

• Purpose of this lab was to:- Study the effect of CSC on human lung cells

- Conduct Microarray analysis of the cells- Analyze the genes/ Compare them with

other Microarray Data- Study one of the genes (PLCB2), and

hypothesize the effect CSC has upon this gene

Methods• Two H1299 cells treated; one with CSC (experimental) and the other as control. • RNA was isolated using Wash Buffers and RNeasy mini-colums.• Then, the cDNA was synthesized and labelled using RNA isolated from control (Cy3)and CSC treated cells (Cy5). RNA was degraded and cDNA was purified.• The labelled nucleotides were added to the cells and hybridized. • The data was analysed via MAGIC Tool Software http://www.dnamicroarray.net/

image/Two-Color%20microarrays.jpg

Results – Down-Regulated GenesTop 20 Down-Regulated Genes

Information for biological processes taken from the NCBI’s gene specific database

Results – Down-Regulated GenesContinued….

Information for biological processes taken from the NCBI’s gene specific database

Results – Down-Regulated Genes

The genes were classified based on their Molecular Function; Binding, Receptor Activity, Catalytic Activity, Transporter Activity and Unknown Function

Molecular Function

BindingReceptor ActivityCatalytic ActivityUnknownTransporter Activity

Results – Down-Regulated Genes

The data was compared to two other students from class and with the pilot data .

PLCB2

NFRKB

PIGR

HTR2B

PPID

GNAT1

SIM2

RFX3

GCNT1

GFRA3

PTRH1

RDH10

ARL8B

LOC145694

SLC25A2

TMC5

MKRN3

E2F2

LMO1

LMLN

-3.5 -3 -2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5

PilotStu2Stu1Exp

Result – Up-Regulated GenesTop 20 Up-Regulated Genes

Information for biological processes taken from the NCBI’s gene specific database

Results – Up-Regulated GenesContinued…

Information for biological processes taken from the NCBI’s gene specific database

The genes were classified based on their cellular function; Channels/Transporters, Involved in Protein Synthesis & Degradation, Involved in DNA Maintenance & Replication, Transcription Factors, Hox Genes and Unknown Function

Cellular Function

Channels/ TransportersProtein Synthesis & DegradationDNA Maintenance & ReplicationUnknownTranscription FactorHox Gene

Results – Up-Regulated Genes

The data was compared to two other students from class and with the pilot data .

CACNA1A

SCG2

C2orf47

COPS2

PQLC1

NR1I2

KLK11

SLC22A6

TMEM27

IL7

CHRNA3

HOXB3

EIF5A

CELA3A

ZNF350

CENTB1

NFYC

ADRA2B

MYOM2

CTNNBL1

-1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 3

PilotStu2Stu1Exp

Results – Up-Regulated Genes

Results

• Major proportion of the down-regulated genes is involved in catalytic activity and binding and few genes in receptor activity

• Major proportion of the up-regulated genes function in protein synthesis and degradation machinery

• Genes appear at various locations for different microarray samples; some are up-regulated whereas others are down-regulated

• Some genes were present in the top 20 of the other list yet some were also missing.

Discussion – PLCB2 Gene

• PLCB2 gene is down-regulated (-3.13)• Full name - Phospolipase beta 2• Model organism – Homo sapiens• Gene type – Protein Coding• Biological Function – Acts as an enzyme

(hydrolyzing phospholipids)

Information for biological processes taken from the NCBI’s gene specific database

Discussion – PLCB2 Gene• Study by F. Brugnoli et al in 20073

•PLCB2 can be regarded as a molecular marker 3

•PLCB2 interaction with actin promotes the association of the phosphoinositide-binding site of PLCB2 with cytoskeleton-associated PIP2 allowing phosphodiesterase activity. 3

Muresan, M. et al. The Loss of Cellular Junctions in Epithelial Lung Cells Induced by Cigarette Smoke Is Attenuated by Corilagin. Oxid Med Cell Longev. Med [Internet]. 2015

Discussion - Hypothesis• Hypothesis : CSC (Cigarette Smoke Condensate) triggers the

release of inhibitors that prevent the interaction between PH domain of PLCB2 gene and actin filaments resulting in loss of migration and cell cycle progression of differentiating cells.

PLCB2

PH Domain

Actin Phospodiester Activity

Cell Migration & Cell Cycle Progression

CSC

Conclusion• CSC contains compounds that

activates inhibitors• These inhibitors prevent the PH

domain of PLCB2 from interacting with the actin filaments

• Prevents the association of the phosphoinositide-binding site of PLC-β2 with cytoskeleton-associated PIP2 hence preventing any phosphodiesterase activity

http://www-tc.pbs.org/wgbh/nova/next/wp-content/uploads/2014/01/lung-cancer-cells-dividing.jpg

References

1. Xiao, Y. et al. Cigarette smoke condensate and individual constituents modulate DNA methyltransferase expression in human liver cells. SAGE Open Med [Internet]. 2015 [Cited 2016 April 5]

2. Muresan, M. et al. The Loss of Cellular Junctions in Epithelial Lung Cells Induced by Cigarette Smoke Is Attenuated by Corilagin. Oxid Med Cell Longev. Med [Internet]. 2015 [Cited 2016 April 5]

3. Brugnoli , F. et al. PLC-beta2 activity on actin-associated polyphosphoinositides promotes migration of differentiating tumoral myeloid precursors. Cell Signal [Internet]. 2007 [Cited 2016 March 30] 1701-12