Introduction cancer cell to be cultured persistently

Introduction

 

Researchers
made a big impact on developing a large number of matching cells to use in
laboratory experiments. There are different cell lines and types, one of the
cell line is which called HeLa cells where human cervical cancer cells found at
the upper top of the vagina and also the entrance to the uterus. It was the initial
human cancer cell to be cultured persistently for experiments and it was
refined in 1951 from Henrietta Lacks.

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HeLa cells develop quickly
when the correct nutrients, disorder and space is used, reason is that HeLa
cells are cancer cells, which can multiply and develop rapidly in a much
uncontrolled way and enters to the normal cells. HeLa cells developed harmful
due to infection with human papilloma virus 18 (HPV18) as HeLa cells can spread
around the body and disturb the normal activity of the cells to make cells convert
cancerous.

In regular
cells, the Hayflick
limit phenomenon is the number of times a
normal human cell will divide until cell division stops. Which means cells can only be
divided by mitosis a specific number of times as the telomeres at the end of
the chromosomes decreases with each division. This doesn’t relate to several
types of cancer cells as they produce an enzyme called telomerase, which
extends the telomeres when chromosomes are copied and permits the cells to
reproduce constantly.

 

 

 

The aim of this experiment is to
test the extracted DNA samples to see whether Arsenic Trioxide (ATO) can affect
HPV18 E6 DNA levels of treated HeLa cells.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Method

 

8
tubes were prepared for this experiment. 7 tubes were labelled 1-6
appropriately and the remaining 1 tube was labelled as NTC. we pipetted
different reagents required for PCR reactions into the PCR tubes which is
stated in the following table.

After
pipetted the reagents, closed the PCR tubes and placed it into the thermocycler
(PCR machine). The following table is showing the thermocycler which was
programmed.

PCR
took approximately 2 hours to complete. Then tubes were collected and stored
under frozen at -20ºC, then we analyzed PCR product
using gel electrophoresis.

 

We used prepared 1% agarose gel containing
8 sample wells.

Electrophoresis buffer gel was
poured in to the tank till the gel was full covered. Then we loaded our samples
(10µL of PCR reactions) into the wells including the 5µL
of the ladder.

Samples were loaded into the wells
by using pipettes then tank was covered with the lid and the power cables were
connected to the power supply (90 Volts).

Started the timer for 30 minutes
after incubation of 30 minutes gel was removed with the tray from the
electrophoresis tank and placed it on a piece of tissue paper then we proceeded
using UV/Dark Reader transilluminator to visualize and analyze  our gel.

 

Results

 

The results obtain are shown in the
figure below and explained in the table-

The
ladder is used to adjust electrophoresis gels
so that samples of unknown DNA that
have been initiated
into the gel
can be measured. HPV18 HeLa and GAPDH – HeLa was untreated which is a template but HPV18 HeLa with 5 µM of ATO was effective and
is seen with a band where it is not as bright as DNA ladder because ATO broke
down its DNA. When ATO concentration increased to 5µM ATO was used for
treatment this was too toxic, therefore all the cell has been destroyed by ATO
so this band is invisible.

GAPDH is one of the key enzymes
involved in glycolysis, also constitutively expressed in almost all tissues in
high amounts. Therefore, GAPDH is widely used as a loading control for protein
normalization and also deregulated in various cancer cells.

 

 

Discussion

 

Arsenic Trioxide (ATO) is a cancer
medication which delays the growth of the cancer cells as well as spreading
around the body. This drug is used to treat acute leukemia, promyelocytic or
APL. General
reports have establish that the anti-proliferative accomplishments of ATO are
mediated by several ways of apoptosis. ATO have been used to treat many cancers
including acute leukemia, promyelocytic or
APL. The molecular bases are not yet stated in solid tumor cells, particularly cervical cancer
cells carrying HPV genome.

The
relation between HPV and cervical cancer is definite then again the treatment
options are limited. The efficient toxicity of ATO has prohibited its extensive
use in medical uses, mainly at the concentrations higher than 5?M as we have
used ATO at low dosage of 2?M which precisely target HPV infected cervical
cancer cells. Where most of the cells were destroyed when we increased the
concentration of ATO to 5?M due to higher drug toxicity, regardless of HPV
infection.

 

Our results showed that HPV18 HeLa (Band
4) with 5µM
concentration of ATO was effective where it is not visible compare to the DNA
ladder, because all the cell has been destroyed by ATO due to high concentration.
HPV18 HeLa (Band 2) was used as
template to compare untreated ATO with treated ATO which is HPV18 HeLa ATO with concentration
of 2µM (Band 3), where I can see that it has effected the cells and its less
visible than untreated HPV HeLa from the results it is showing that the treatment
on the cancer was as successful as I was expected. GAPDH (Band 8) was untreated
as it was a template also. GAPDH is used as loading control for protein to normalize
and deregulate in several cancer cells. As GAPHD is an enzyme which involves in
glycolysis constitutively expressing in majority tissues in high amounts. Band 4,
5, and 6 are DNA extra used as template, this was used as an internal control.

 

 

 

Conclusion

 

 

From my results I can conclude that ATO
has affected HPV18 E6 DNA levels as there is a strong evidence that ATO
restrain proliferation and induced apoptosis to the HeLa cells showing direct
effects on cervical cancer. As ATO considerably
decreased cell in comparison with
control cells in a time and does dependent method. At the highest concentration
(5µM),
ATO inhibited the progression prospective of the cell proliferation which
caused a rapid decline in the number of visible cells after 2 days, there was
no visible cells remained (HPV18 HeLa ATO 5 µM band 4), at the lowest
concentration (2µM) cell proliferation was initially increased as we compared
with control, and had small effect (HPV18 HeLa ATO 2 µM band 3). High concentration
and low concentration had no effect on the HPV18 HeLa Untreated band 2 and
GAPDH bands.