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DEFINING THE ROLE OF MUTATED Hbraf
IN MELANOGENESIS
EUNICE ASARE
Dr. ALAN HOUGHTON LABORATORY
Memorial Sloan Kettering Cancer Center
(MSKCC)
INTRODUCTION

Melanoma is the deadliest skin cancer. It begins
in melanocytes (cells responsible for skin
pigmentation).

There is a frequency of Braf mutations in ras
pathway found in most cases of melanoma,
~7O%.

Braf is a protein coding gene that enables a
normal cell division.
RISK FACTORS LEADING TO
MELANOMA
CHANGING NEVUS(MOLE)
 LARGE NUMBERS OF MOLES
 FAMILY HISTORY OF MELANOMA
 OVEREXPOSURE TO SUN
ULTRAVOILET RADIATION
 MUTATION OF GENE (Hbraf)

CHARACTERISTICS OF MELANOMA
Pathways that may lead to cell cycle dysregulation in melanocytes
CELL CYCLE
Ras
Pten
CdKn2a
locus
Process by which a cell divides to
Form a new cell.
The gene product of braf interacts with other gene products in the cell for a normal cell
division.
A mutation in the pathway will cause the cell to divide abnormally, leading to cancer.
There is a frequency of Braf mutations in ~70 of most cases of Melanoma.
OBJECTIVE
Generation of transgenic mice expressing
the mutated Hbraf in melanocytes to mimic
the situation in human melanoma patients
with mutated Hbraf
-
Insertion of mutated Hbraf into expression vector
Expression of mutated Hbraf in melanocytes
Use of transgenic mice to express mutated Hbraf
Create transgenic mice susceptible in developing
melanoma.
Vaccinate mice who develop melanoma with
HGP75 optimized or HGP75 vaccine.
STRATEGY TO INSERT Hbraf INTO EXPRESSION VECTOR
MATERIALS and METHODS

PLASMID CONSTRUCTION : The oligo TRP2 1
Braf 1 and TRP2 Braf 2 is prepared from a sequence of DNA which
contains the restriction sites for Xhol, NoTI, XbaI, HindIII, BAMHI to
be ligated into the original plasmid.

INSERT OF TRP2: An obtained plasmid pPtrp-2/lacZ
has the Hbraf gene promoter Trp-2. To attain only the Trp-2, we
cleaved the plasmid of its other genes and promoters by digesting
using NoTI.
continued
Plasmid vector pPtrp-2/lacZ containing the Hbraf
gene promoter Trp-2.
CONTINUED




INSERT OF Hbraf. The final insert into our plasmid is
Hbraf wild-type and Hbraf mutated. An acquired plasmid
pcDNA 3.1(-) with a size of 5427bp contains both insert
but also with other genes.
we only want the wt and mut Hbraf genes. We
digested the plasmid with PmeI, next agarose gel
electrophoreses was run to check for the purity of the
insert.
Two separate ligations are done here; the first one is to
insert the mut Hbraf into our constructed plasmid and
the next ligation is to insert the wt Hbraf into our
constructed plasmid without the mut Hbraf.
Transformation is prepared with both newly constructed
plasmids thus 62.1mod/oli/Trp2+mutH and
62.mod/oli/Trp2+wtHbraf.
DESIGNING TRANSGENIC MICE
FUTURE DIRECTIONS
• In the course of the coming weeks our ongoing objectives will be
headed for and completed. Since we have already constructed our
expression vector with the desired gene mutHbraf, it will be
expressed in melanocytes.
• In the direction of inserting the mutHbraf in melanocytes, it will be
sent to the Transgenic Mice Facility at MSKCC for this to be done.
• The Embryonic Stem Procedure will be used to create a line of
transgenic mice C57B/6 vulnerable in developing melanoma
comparable to human melanoma patients with mutated Hbraf gene.
• The C57B/6 mice will be tested for the presence of mutBraf gene.
• The mice that test out positive for the gene will be followed to see if
they develop melanoma.
• The melanomas developed will be characterized and treated with
the HGP75 optimized or HGP75 vaccine to suppress the melanoma.
GENOTYPING TRANSGENIC
MICE
Presently in the lab our ongoing aim is to:

Use Polymerase Chain Reaction (PCR) method to detect the presence of
MutHbraf in our obtained transgenic mice.

We will cross breed MutHbraf transgenic mice with mice whose Ink4a
gene ( tumor suppressor gene) has been “Knocked Out”.

New mice breed from the cross will be monitored to see if they develop
melanoma.

The melanomas developed will be characterized and treated
with the HGP75 optimized or HGP75 vaccine to suppress the
melanoma.
Pathways that may lead to cell cycle dysregulation in melanocytes
Pten
Ras
CdKn2a
locus
GENOTYPING TRANSGENIC
MICE
Presently in the lab our ongoing aim is to:

Use Polymerase Chain Reaction (PCR) method to detect the presence of
MutHbraf in our obtained transgenic mice.

We will cross breed MutHbraf transgenic mice with mice whose Ink4a
gene ( tumor suppressor gene) has been “Knocked Out”.

New mice breed from the cross will be monitored to see if they develop
melanoma.

The melanomas developed will be characterized and treated
with the HGP75 optimized or HGP75 vaccine to suppress the
melanoma.
AKNOWLEDGEMENT

ELVA BARREDA and Dr TAHA MERGHOUB

MINYI TAN

SHELDON MOORE

BARTEK JABLONSKI
 ALAN HOUGHTON
LABORATORY OF IMMUNOLOGY

MSKCC
 Dr. SAT BHATTACHARY
HARLEM CHILDREN SOCIETY
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REFERENCE
(1) (1)MouseGENETICSANDTRANSGENICS
THE Practical Approach SERIES; B.D Hames
http:www.oup.co.uk/PAS
(2) Houghton AN, Polsky D. Focus on melanoma. Cancer Cell. 2002 Oct;2(4):275-8.
Review
(3) Tuveson DA, Weber BL, Herlyn M. BRAF as a potential therapeutic target in
melanoma and other malignancies. Cancer Cell. 2003 Aug;4(2):95-8. Review.
(4) JK. Related Links Is there more than one road to melanoma?
Lancet. 2004 Feb 28;363(9410):728-30. Review.
PMID: 15005091 [Pub Med - indexed for MEDLINE
(5) Immunity to cancer through immune recognition of altered self: Studies with
melanoma. Jose A. Guevara-Patino, Mary Jo Turk, Jedd D. Wolchock, and Alan N.
Houghton
MSKCC and the Weill Graduate school of Medical sciences and Medical school of
Cornell University, 1275 York Avenue, New York, N.Y 10021
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HELEN DAVIES et al
Cancer Genome project, The welcome Trust Sanger Institute Welcome Trust Genome
Campus, Hinxton CB10ISA,UK et. al
(7)American Cancer Society Web site; what are the key statistics for melanoma skin
cancer?
http://www.cancer.org
(8) Cancer Reference Information
skin cancer –Melanoma
what are the risk factors for melanoma?
http:www.cancer.org/docroot/cri/content/cri_2_4_2x what are the risk
Melanoma
Escape dangers by learning to protect yourself
http://www.ftmeade.army.mil/soundoff/archives/SO2002/Jul18/html/Cover-story.htm
Incidence of Braf oncogene mutation and clinical relevance for Primary Cutaneous
Melanomas
Masaru Shinozaki et. al
Department of Molecular oncology, John Wayne cancer institute, Saint John’s Health
Center, Santa Monica, California