B16 F10 melanoma: an investigation of genetic stability
Erica Martin, advised by Dr. Spilatro
Introduction
Melanoma is a type of skin cancer that is becoming increasingly common. In
recent years, rates of increase in melanoma cases among Caucasian have been
between 3-7% per year [1] . Additionally, the prevalence of melanoma doubles
every 10-20 years [1]. Melanoma is able to spread to other parts of the body,
through the process known as metastasis. One method used to study
melanoma is the murine B16 melanoma cell line.
Chromosomal aberrations of various types cause melanoma. Normal
C57BL/6J mice have 40 chromosomes [2], whereas the B16 melanoma cell line
has a variable number of chromosomes ranging from 68 to 77 [3]. The
variability in the B16 melanoma cell line is something that must be considered
when using the cell line to make generalizations about human cancers: is the
B16 melanoma cell line a good model for human melanoma? To study
chromosomal abnormalities, a karyotype can be used. A karyotype is a pictorial
representation of the chromosomes in a cell. The karyotype can be used to
determine various characteristics of a cell line, ranging from chromosome
number to changes in individual chromosomes. My research is focused on
determining if there will be increased karyotypic abnormalities in the B16 F10
cell line as compared to representative normal C57BL/6J mouse cell line. My
hypothesis is that there will be more chromosomal abnormalities in the B16 cell
line as compared to representative normal C57BL/6J mouse cell line.
To produce a karyotype, chromosome spreads can be used as a method of
visualizing mitotic chromosomes. There are various factors that affect the
quality of chromosome spreads, including the height from which cell
suspensions are dropped. Once a quality chromosome spread is obtained, the
chromosomes can be G-banded to produce visible banding. This banding allows
pairing of homologous chromosomes, which have distinctive banding patterns.
The G-banding protocol must be adapted to the organismal origin of a
chromosome spread, the age of the chromosome spread, and other factors.
Methods
Cell culturing
B16 F10 melanoma cells were grown in CDMEM growth medium for 3-5
days and then released from the bottom of the culture flask with trypsin. The
growth medium contained dialyzed FBS, DMEM with L-glutamine, and penicillin
streptomycin antibiotic. The cell suspension was then added to a new culture
flask with fresh growth medium.
Preparation of chromosome spreads
Cultured cells in the mid-log phase of growth (when the cells are dividing
most rapidly) were treated with Demecolcine (0.0366 ug/mL) in CDMEM for
varying amounts of time, causing actively dividing cells to arrest in prophase.
The cells were then released from the plate with trypsin, and the cell
suspensions were incubated at 37°C for 20 minutes with hypotonic citrate
solution [5]. Chilled acetic MeOH was added, and the suspension was
centrifuged. The supernatant was discarded and the remaining pellet was
gently vortexed while chilled acetic MeOH was added to the pellet. The cell
suspension was then left on ice for 10 minutes, and resuspended in fresh acetic
MeOH. The cell suspensions were then dropped from a height of approximately
16 inches onto chilled microscope slides. The slides were then passed over a
boiling water bath, as steam was suggested to facilitate further spreading [6].
The slides were then air dried.
G-banding of chromosomes
Chromosome spreads were treated with a solution of 0.85% NaCl
(working salt solution) and trypsin, which partially digested portions of the
chromosomes [4]. The trypsin concentration and treatment time with the
solution was varied. The spreads were then stained with a solution of
Giemsa stain and Gurr buffer and allowed to air dry.
Conclusions
Figure 1: The apparatus that was used
for dropping cell suspensions. The
suspension was taken up into a Pasteur
pipet and the pipet was secured in the
clamp on the apparatus. A chilled slide
was placed directly beneath the pipet
on the lab bench and the cell
suspension was dropped onto the slide.
Figure 2: A representative
chromosome spread, with an
intact nucleus to the left of the
chromosome spread.
The chromosome number of a C57BL/6J mouse is 40,
whereas the average number of chromosomes that I found in
the B16 F10 cells was 74.6 [2]. The mean chromosome
number found by Kendal et al. was 73 [3]. My results ,
supplemented by those of Kendal et al. suggest that there is a
substantial amount of aneuploidy in the B16 F10 cell line.
Chromosomal aneuploidy is defined as having extra
chromosome as a result of aberrant mitotic processes.
Because there was a fairly large variance in the number of
chromosomes in the B16 F10 chromosome spreads, this is
indicative of the relative variability of the karyotype between
generations of the cultured cells. This outcome is to be
expected, as it is well known that chromosomal aberrations
are propagated and accumulated in cultured cell lines.
The purpose of my research was to determine if there are
increased karyotypic abnormalities in the B16 F10 cell line as
compared to representative normal C57BL/6J mouse
karyotypes. The chromosomes were matched cursorily, as the
somewhat faint banding did not allow for definitive
chromosome pairing. The karyotype that I produced did not
allow for analysis of specific chromosomal abnormalities.
Literature Cited
Figure 3: A representative Gbanded chromosome spread
obtained from the G-banding
procedure.
Figure 4: The G-banded
karyotype resulting from
manipulation of the
chromosome spread in Figure 3.
Results
Figure 2 shows an image of a nonmitotic nucleus and indicates that
chromosomes can be seen without the trypsin treatment used in the Gbanding protocol. In this image, the chromosomes are stained and spread in
preparation for obtaining a karyotype.
Figure 3 shows an image of the results of the G-banding procedure. In this
image, the chromosomes are banded fairly well. Figure 5 shows an image of
a karyotype obtained from the G-banding procedure. There are 75
chromosomes in this particular spread.
To determine the number of chromosomes that is representative of the
B16 F10 cell line, I counted the number of chromosomes in 20 randomly
selected chromosome spreads . The range of the chromosome number in
the spreads that I counted was from 70 chromosomes to 80 chromosomes. I
then computed the mean and standard deviation of this data, and found the
average number of chromosomes was 74.7, with a standard deviation of
3.56.
[1] Lens M, Dawes M. 2004. Global perspectives of contemporary
epidemiological trends of cutaneous malignant melanoma. British Journal
of Dermatology 150: 179-185.
[2] Crippa M. 1964. The mouse karyotype in somatic cells cultured in
vitro. Chromosoma 15: 301-311.
[3] Kendal WS, Wang R, Hsu TC, Frost P. 1987. Rates of generation of major
karyotypic abnormalities in relationship to the metastatic potential of B16
murine melanoma. Cancer Research 47: 3835-3841.
[4] Veile R. Method: staining chromosomes (G-banding). [Internet]
Department of Genetics at Washing ton University in St. Louis School of
Medicine. [created 1990 May 21; cited 2011 Apr 12]
(http://humgen.wustl.edu).
[5] Freshney, RI. 1994. Culture of animal cells: a manual of basic technique,
4th. ed. New York (NY):Wiley-Liss; (577).
[6] Henegariu O, Heerema NA, Wright LL, Bray-Ward P, Ward DC, Vance GH.
2001. Improvements in cytogenetic slide preparation: controlled
chromosome spreading, chemical aging, and gradual denaturing.
Cytometry 43: 106.
Acknowledgements
I would like to thank Dr. Spilatro, my research advisor, for his
patient and encouraging guidance throughout the duration of
my research. Additionally, I would like to thank the Marietta
College Investigative Studies committee for funding my
summer research. Dr. Brown was helpful in offering valuable
insight on the presentation of my research. I also appreciate
the entire biology department’s involvement and
encouragement with my research, especially with regards to
funding. The input that my capstone classmates offered in
perfecting my presentation was also useful. Last but not least, I
thank my family and friends who have offered unending
support and encouragement.