ABSTRACT

Cancer is a potentially fatal disease caused partly by genetic changes and partly by
environmental factors that mutate genes that encode critical cell-regulatory proteins. The
resultant aberrant cell behaviour leads to expansive masses of abnormal cells that destroy
surrounding normal tissue and can spread to vital organs resulting in disseminated disease,
commonly a harbinger of imminent patient death .

Background: It is estimated that about 4.5 million people die from cancer each year in the world.
Cancer is the uncontrolled growth of abnormal cells in the body. Cancer can develop in almost
any organ or tissue, such as the lung, colon, breast, skin, bones, and central nervous system
(CNS).

The prognosis for patients with cancer metastases to brain is extremely poor. Large-conductance
calcium- and voltage-activated potassium channel (BKCa) channels are significantly upregulated
in different cancer cells. These channels re also im0licated in cancer cell proliferation, migration
and invasion and growth.

Methodology: In this study we have investigated the role of KCNMA1 gene that encodes for the
pore-forming α-subunit of BKCa channels, in primary and metastatic cancer cell lines acquired
from ATCC (USA). Single strand (Ss) cDNA will be generated using the mRNA extracted from
the cells or tissues, and then KCNMA1 expression was checked by RT-PCR and Agarose gel
electrophoresis.

Outcome: The present study will help us to identify the expression of KCNMA1 gene in different
types of cancer. This data will be helpful to decide on the types of cancer that can over-express
the gene and should be therapeutically treated by blocking the KCNMA1 gene.

The expression levels of KCNMA1 in cancer cells varied from one cell type to the other cell
type, although the expression levels was qualitative. On the other hand, the KCNMA1
expression was greater in cancer cells than in normal cells.

In conclusion, based on the experimental observation we conclude that cancer cells over express
KCNMA1 compared to normal cells. Hence KCNMA1 appear to promote cancer cell growth.
Future research will explore the actual role of this K+ channels in cancer progression,
development and growth using cancer tissues obtained from patients.
 CONTENTS

SL NO TOPICS PAGE NO

1 INTRODUCTION 1

2 LITERATURE SURVEY 15

3 REQUIREMENTS 24

4 METHODOLOGY 26

4.1 STEPS FOR PRIMER DESIGNING 27

4.2 CELL LINES WORKED UPON 32

4.3 RNA EXTRACTION AND PCR 34

4.4 PROTOCOL FOR RNA ISOLATION 34

4.5 PCR 35

4.6 PROTOCOL FOR cDNA PREPARATION 39

4.7 AGAROSE GEL ELECTROPHORESIS 40

4.8 IN SILICO STUDIES 41

5 RESULTS 43

5.1 PRIMER DESIGNING 44

5.2 CELL LINES WORKED UPON 45

5.3 RNA EXTRACTION 46

5.4 cDNA AND PCR PRODUCT 47

5.5 GEL ELECTROPHORESIS RESULTS FOR KCNMA1 AND 50

GAPDH GENE

5.6 RESULTS OF IN SILICO STUDIES 53

6 DISCUSSION 58

7 CONCLUSION 62

8 REFERENCES 64
 9 WEBLINKS 68

LIST OF TABLES

SL NO TOPICS PAGE NO

1 LIST OF ION CHANNELS AND THEIR ACTIVITY 7

2 CELL LINES OBTAINED FROM ATCC 40

3 THE REAGENTS USED FOR THE AMPLIFICATION OF 32

cDNA

LIST OF FIGURES
SL NO TOPICS PAGE NO

1 A) MULTISTAGE CARCINOGENESIS FROM THE 5

GENETIC PERSPECTIVE

B) THE CONSEQUENT MALIGNANT PHENOTYPE

2 PCR CYCLE 12

3 NCBI ENTREZ-CROSS SEARCH DATABASE 27

4 mRNA SEQUENCE OF KCNMA1 GENE 28

5 PRIMER3’s WWW INPUT PAGE WITHOUT USER INPUT 31

6 PRIMER3’s WWW INPUT PAGE AFTER THE USER HAS 32

ENTERED “SEQUENCE ID,” AND A “TARGET.”

7 THE PCR SYSTEM 2700 34

8 THE CONTOL PANEL FOR THE PCR SYSTEM 2700 36

9 GRAPHICAL REPRESENTATION OF A METHOD, ON THE 37

CREATE/EDIT SCREEN

10 SAMPLE OUTPUT FOR PRIMERS KCNMA1 & GAPDH 44

GENES

11 IMAGES OF CELL LINES USED 45

12 RNA WAS EXTRACTED FROM THE 12 ATCC CELL LINES 46

USING TRIZOL REAGENT

13 THE RNA OBTAINED FROM THE CELL LINES WAS USED 48

TO MAKE cDNA, WHICH WAS USED TO CARRY OUT
FURTHER PCR REACTIONS

14 GEL ELECTROPHORESIS RESULT FOR KCNMA1 GENE 50

15 GEL ELECTROPHORESIS RESULT FOR GAPDH GENE 51

16 DERIVED STRUCTURE OF KCNMA1 GENE 53

17 Q- SITE FINDER ONLINE PREDICTION 54

18 MINIMIZATION OF LIGANDS 55
19 SNAPSHOTS OF DOCKING PROCESS 56

20 AMES TEST 57