Download Investigation of the role of the ras protooncogene point mutation in

Investigation of the Role of the ras Protooncogene Point
Mutation in Human Uveal Melanomas
Charles N. Soparker,* Joan M. O'Brien,^ and Daniel M. Albertf
Purpose. Genetic alterations have been observed in a wide variety of neoplaslic processes,
including Burkitt's lymphoma, chronic myelogenous leukemia, promyelocytic leukemia, and
solid tumors of the colon, skin, and breast. The polymerase chain reaction (PCR), dot blotting,
and direct double-stranded DNA sequencing were used to assess ras gene activation in human
uveal melanomas for three candidate genes: c-Ha-?a.s], c-Ki-ras2, and N-ras at codons 12, 13,
and 61.
Methods. Samples of 49 human uveal melanomas were obtained. Amplifiable high molecular
weight DNA was obtained from 39 of these. PCR amplification of regions centering on three
candidate ras genes was performed. PCR-amplified DNA was evaluated by dot blot and doublestranded DNA sequencing utilizing standard methods.
Results. No point mutations were identified in screening the c-Ha-ras gene nor were any genetic alterations found in the c-Ki-ra.?2 gene at codons 12 and 13. Only wild-type sequences
were found at codon 61. No ras mutations were detected in any uveal melanomas studied.
Conclusions. This study provides no evidence to support an association between ras protooncogene mutations and human uveal melanomas at codons 12, 13, or 61. Invest Ophthalmol Vis
Sci. 1993;34:2203-2209.
irotooncogenes are naturally occurring genes that
influence cellular growth and differentiation.1"4 Gene
amplification, point mutation, or DNA rearrangement
occurring at such important regulatory sites may result in protooncogene "activation" and subsequent
instigation of neoplastic change. Unique relationships
have been observed between specific protooncogenes
and such neoplastic states as Burkitt's lymphoma,
chronic myelogenous leukemia, promyelocytic leukemia, colonic cancer, epidermal cancer, and breast
cancer.5"9
From the *Departm<mt of Pathology, University of Massachusetts Medical Center,
Worcester, and the iDavid G. Cogan Eye Pathology Laboratory, Massachusetts Eye
and Ear Infirmary, Boston, Mtussachmelts.
Supported by National Eye Institute (Hethesda, MD) grant EY0I917 (DMA).
Submitted for publication: Jamiary 15, 1992; accepted January 15, 1992.
Proprietary interest allegory: N.
Reprint requests: Daniel M. Albert, MD, University of Wisconsin - Madison,
Department of Ophthalmology, F4 334 Clinical Science Center, 600 Highland
Avenue, Madison, Wl 53792-3220.
Among the protooncogenes, the ras family of
three highly homologous genes (c-Ha-rasl, c-Ki-ra?2,
and N-ras), which share near identity with two virally
carried genes (c-Ha-ras and c-Ki-r&y) and a significant
sequence homology with the rho (ras homologue)10
genes and the genes encoding the alpha subunits of
the transmembrane signal-transducing G proteins. It
appears that the three ras genes are activated at different frequencies in different tumor types. In bladder
and urinary carcinomas, c-Ha-r&sl is most commonly
activated,1112 whereas in lung, pancreatic, and colonic carcinomas c-Ki-ra?2 mutations are more frequent.13"18 N-ras activation predominates in hematopoietic malignancies and also perhaps cutaneous malignant melanomas.19"23
In this study, polymerase chain reaction
(PCR),2*25 dot blotting,23 and direct double-stranded
DNA sequencing techniques were used to attempt to
identify alterations in the base sequences in c-Hz-rasl,
Investigative Ophthalmology & Visual Science, June 1993, Vol. 34, No. 7
Copyright © Association for Research in Vision and Ophthalmology
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Investigative Ophthalmology & Visual Science, June 1993, Vol. 34, No. 7
2204
c-Ki-ras2, and N-ras protooncogenes in 53 human
uveal melanomas and 6 cutaneous melanomas at codons 12, 13, and 61. These codons were selected because of the finding that other neoplasms, including
cutaneous melanoma, have been associated with activating mutations at these sites.
cutaneous melanomas. Of the 43 uveal melanomas
used to prepare high molecular weight DNA, 19 were
from women and 24, from men. The ages of patients at
the time of enucleation ranged from 25-87 yr (mean,
61.7 yr; median, 62 yr). Forty of the melanomas were
of the mixed cell type, one was of the epithelioid cell
type, and two were of the spindle cell tumors.27
MATERIALS AND METHODS
PCR Amplification and Fragment Purification
The tenets of the Declaration of Helsinki (published in
May 1992) were followed. Institutional human experimentation committee approval as exempt (pathologic
material) was granted for this research.
PCR selective amplification of regions centering on
codons 12, 13, and 61 of c-Ha-rarl, c-Ki-ras2, and Nras was performed using a modification of a method
previously described.29 Briefly, 200-300 ng of high
molecular weight genomic DNA was isolated and protease inactivated by heating at 95°C for 5 min. This
DNA was added to 50 n\ of a reaction mixture containing one unit of Taq polymerase (Perkin Elmer-Cetus,
Norwalk, CT), 50 mmol/1 KC1, 2 Mg/M' bovine serum
albumin, 20 mol/1 Tris, 200 /amol/1 of each of the four
nucleotide triphosphates (Pharmacia, Piscataway, NJ),
and single-stranded oligonucleotide primer pairs that
were synthesized and gel purified in our laboratory or
obtained from Clontech (Palo Alto, CA). The reaction
mixtures were layered with 40 /A of mineral oil (Fisher
Scientific, Fairlawn, NJ) to prevent evaporation during heating. The specific pH, Mg++ concentration,
and primer pair concentrations are listed in Table 1
for each protooncogene region amplified. All amplification reactions were conducted using a thermal
cycler (EriComp, San Diego, CA). The primary denaturation was performed at 93°C for 20 sec. The final
annealing and polymerization were conducted for 1.5
and 4 min, respectively.
Isolation of Melanoma High Molecular Weight
DNA
Forty-nine uveal melanomas were obtained from the
enucleated eyes of patients enrolled in the Collaborative Ocular Melanoma Study (COMS),26 and they were
classified histopathologically by one of the authors
(DMA) at the Massachusetts Eye and Ear Infirmary,
according to a modification of the Callender classification system.27 The use of COMS tissue for this study
was approved by the COMS Executive Committee. An
additional four specimens from uveal melanomas were
submitted to the David G. Cogan Eye Pathology Laboratory from sources outside the COMS and similarly
examined by light microscopy. Histopathologically
diagnosed primary cutaneous malignant melanomas
of the nonlentigo malign a type were obtained from six
consecutive patients of a single dermatologist. The
largest portion of each tissue sample was used for histopathologic diagnosis, but tumor fragments of 5—10
mm3 were taken for DNA isolation. Histologic sections
were evaluated to ensure that the tumor fragments
contained minimal amounts of contaminating normal
stroma or inflammatory cells. The tumor fragments
were stored at —70°C or — 196°C, and isolation of
high molecular weight DNA was accomplished using
standard techniques.28
Amplifiable high molecular weight DNA was isolated from 39 of 49 uveal melanoma tumor fragments
received from COMS participants. Adequate DNA for
amplification of at least one ras gene was available
from four additional uveal melanomas and from six
After PCR amplification, the reaction mixtures
were extracted with an equal volume of chloroformisoamyl alcohol (24:1 vol/vol) to remove the mineral
oil, incubated for 1 hr at 37°C with 500 fig/m\ of proteinase K (Boehringer Mannheim, Indianapolis, IN) to
cleave DNA-bound proteins, extracted again with an
equal volume of phenol-chloroform (1:1 vol/vol), and
then extracted again with an equal volume of chloroform-isoamyl alcohol (24:1 vol/vol) to remove the
phenol. The samples were diluted to 1.5 ml with sterile
water and then concentrated and purified away from
the 20-base pair single-stranded oligonucleotide
TABLE l. PCR Amplification Conditions
Genomic
Region
K' 12,1S
Kisi
Ha ]2 13
Ha61
N,2,IS
NB,
Annealing
Temperature
CO
58
50
60
60
56
56
MgCl2
(mmol/l)
pH
5' Primer
(pmol)
3' Primer
(pmol)
2.50
1.50
1.00
1.00
2.00
1.75
8.4
8.5
8.4
8.4
8.4
8.4
20
40
20
20
20
20
20
20
20
20
20
20
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Investigation of ras Protooncogene
2205
TABLE 2. ras Gene Primer Sequences
Genomic
Region
Ki6,
Ha|2,i3
Ha 6 |
N l2 ,13
Amplified Fragment Length
(bp)
Primer Sequences
5'-ATGACTGAATATAAACTTGT
3'-CTCTATTGTTGGATCATATT
5'-AAGTAGTAATTGATGGAGAA
3'-AGAAAGCCCGCCCCAGTCCT
5'-ATGACGGAATATAAGCTGGT
3'-CGCCAGGCTCACCTCTATA
5'-AGGTGGTCATTGATGGGGAG
3'-AGGAAGCCCTCCCCGGTGCG
5'-ATGACTGAGTACAAACTGGT
3'-CTCTATGGTGGGATCATATr
5'-CAAGTGGTTATAGATGGTGA
3'-AGGAAGCCTTCGCCTGTCCT
primers by centrifugation in Ventricon-30 columns
(Amicon Grace, Danvers, MA) using a SS fixed-angle
rotor (Sorvall, E. I. DuPont de Nemours, Wilmington,
DE) at room temperature at 5000 X g for 20 min. The
final concentrates were evaporated dry and then resuspended in 50 jd of 10 mmol/1 Tris HC1 (pH 7.5) and 1
mmol/1 ethylenediaminetetraacetic acid (EDTA).
Then 2 pi of this final solution was analyzed for purity
and approximate quantitation by gel electrophoresis
in 2% agarose (International Biotechnologies, New
Haven, CT).
5'-Phosphorus-32 Labeling of Oligonucleotide
Probes
Single-stranded oligonucleotides representing all possible point mutations at each of the nine pertinent raj
genetic locations were obtained from Clontech and
were end labeled with 7-phosphorus-32-adenosine triphosphate, using a modification of previously described methods.28 We heated 100 ng of each oligonucleotide representing a putative mutation at a single
ras genetic location to 60°C for 5 min to allow full
denaturation. The oligonucleotides were phosphorylated using T4 polynucleotide kinase (US Biochemicals, Cleveland, OH) with 5 ^1 of 7-phosphorus-32-labeled adenosine triphosphate (specific activity, 6000
Ci/mmol/1; New England Nuclear, Wilmington, MA)
by incubation for 20 min at 37°C in a final volume of
50 ^1 in 50 mmol/1 Tris HCI (pH 7.5), 10 mmol/1
MgCI2, 5 mmol/1 dithiothreitol, and 0.1 mmol/1 spermidine. Labeled probes were then purified from contaminant free 7-phosphorus-32-labeled adenosine triphosphate by gel filtration.28 They were evaporation
concentrated and examined for purity by thin-layer
chromatography in 0.3 mol/l KPO4 (pH 7.5).
Dot Blotting
Then 45 ix\ of tumor fragment PCR-amplified DNA
(prepared as described) was brought to a volume of
475 M! (in 400 mmol/1 NaOH and 25 mmol/1 EDTA)
heated at 95°C for 2 min and then placed on ice for 10
111
109
117
109
111
110
min after addition of 475 MI of 4°C Tris HCI, pH 7.4.
Dot blotting using a dot blot manifold (Schleicher and
Schuell, Keene, NH) was performed by applying 100
lA of each DNA solution to Zeta-probe membranes
(BioRad, Richmond, CA) with a vacuum applied. Each
well was rinsed twice with 200 jul of 20X SSPE (containing 20 mmol/1 EDTA, 3 mol/l NaCl, and 200
mmol/1 NaH2PO4, pH 7.4). The filters were then
baked at 80°C under vacuum for 4 hr. Nine identical
blots were carried out in 5X SSPE and 5X Denhardt's
(containing 0.1% Ficoll (Sigma Chemical, St. Louis,
MO) 400, 0.1% polyvinylpyrrolidone, and 0.1% bovine serum albumin) with 0.5% sodium dodecyl sulfate
and 100 mmol/1 sodium pyrophosphate, pH 7.5, for 2
and 12 hr, respectively. Hybridization was were performed through addition of either a wild-type ras sequence probe at a final concentration of 5 X 106 cpm/
ml or a mixture of mutant sequence probes for a specific ras genetic location, each at a concentration of 5
X 10 6 cpm/ml.
After hybridization, a preliminary wash was performed at room temperature for 20 min using 1 mol/l
NaCl and 0.1 mol/l sodium citrate (pH 7.0), followed
by another wash at room temperature for 20 min in
Solution T (3 mol/l tetramethyl ammonium chloride,
50 mmol/1 Tris HCI [pH 8.0], 2 mmol/1 EDTA, and
0.1% sodium dodecyl sulfate). The filters were then
washed twice in a shaking water bath at 63°C for 40
min in Solution T. A final rinse was performed in 1
mol/l NaCl and 0.1 mol/l sodium citrate (pH 7.0). The
blots were exposed to XAR film (Eastman Kodak,
Rochester, NY) at —70°C with intensifying screens for
0.5 and 24 hr.
Filters hybridized with putative mutant probes
were heated in 0.1% sodium dodecyl sulfate and 5
mmol/1 EDTA (pH 8.0) at 80°C for 60 min, autoradiographed to ensure all radioactive labels had been
stripped, and rehybridized with wild-type probe.
Direct Double-Stranded Sequencing
Direct bidirectional sequencing of double-stranded
PCR-amplified DNA was performed through modifica-
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Investigative Ophthalmology & Visual Science, June 1993, Vol. 34, No. 7
2206
tion of standard phosphorus-32-labeling methods.30
Samples for direct sequencing were selected randomly
to confirm dot blot analysis. Identical primers used for
PCR amplification were used for sequencing the approximately 100-base pair PCR products in both directions. We end labeled 40 ng of primer with 30 /tCi
phosphorus-32 using T4 polynucleotide kinase as described. This was ethanol precipitated and resuspended in 7 yul of water. The primer and PCR-amplified template (1:10 vol/vol) were used to initiate T7
DNA polymerase activity (US Biochemical). Sequence
analysis was performed on an 8% polyacrylamide sequencing gel.28
RESULTS
Ten of the 53 uveal melanomas (23.3%) could not be
amplified (seven mixed cell and three spindle cell) and,
therefore, were dropped from the study. Not all tumor
fragments yielded undegraded, high molecular weight
genomic DNA amenable to amplification at all nine ras
genetic locations (Table 3). No point mutations were
found in screening the c-Ha-rasl gene of 23 uveal melanomas, nor were any genetic alterations found in the
c-Ki-ras2 gene at codons 12 and 13 of 36 uveal melanomas or at codon 61 of the 39 uveal melanomas.
Similarly, investigation of the N-ras gene at codons 12
and 13 of 33 uveal melanomas revealed only wild-type
sequences. Figure 2 shows representative dot blot analysis of three uveal melanomas and one cutaneous melanoma. Of six cutaneous melanomas, only one showed
a point mutation at codon 12 of the N-ras gene. This
malignant melanoma contained no wild-type sequences for the N-ras gene at codon 12. Direct sequence analysis of this mutation revealed guanine to
adenine substitution, which would result in a glycine to
aspartate change in the protein product. Random partial or complete bidirectional sequence analysis of the
domains of interest in nine other melanomas confirmed the dot blot analysis.
TABLE 3. ras Gene Locations
Ki| 2
Ha,
N 12
N,,
Ki13
Ha,
GGAGCT
GGCGCCGGC
GGAGCA
GGAGCAGGT
• • •
• • •
• • •
• • •
GGT
AGT
CGT
TGT
GAT
GCT
GTT
GGAGCTGGT • • •
GGCGCC • • •
GGC
AGC
CGC
TGC
GAC
GCC
GGCGTAGGCAA
GTGGGCAA
GGTGTTGGGAA
GTTGGGAA
g'y
Wild-type
ser
arg
cys
asp
ala
val
ACAGCAGGT • • • GAGGAGTA
ACAGCTGGA • • • GAAGAGTA
CAA
Ha 61
Wild-type
GTAGGCAA
GGTGTGGGCAA
GTC
Wei
gly
ser
arg
cys
asp
ala
val
GAA
CCA
CGA
CTA
CAC
CAT
ACCGCCGGC • • • GAGGAGTA
CAG
AAG
GAG
CCG
CGG
CTG
CAC
CAT
gin
glu
pro
arg
leu
his
his
Wild-type
gin
lys
glu
pro
arg
Wild-type
leu
his
his
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Investigation of ras Protooncogene
„
#
AGE
(vrs)
1
2
3
4
5
6
7
8
9
10
59
71
84
54
81
78
62
36
87
64
SEX
12
13
14
15
16
17
18
73
68
58
37
41
34
61
F
M
M
M
F
F
M
M
F
M
F
F
F
M
F
M
F
M
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
1C
2C
3C
4C
5C
6C
82
60
62
62
68
81
25
62
74
70
46
58
64
66
43
64
60
67
69
56
54
60
61
66
F
F
M
M
M
F
F
F
F
M
F
M
F
F
M
M
M
M
F
M
M
M
M
M
1 1 5 5
2207
CELL
TYPE
I
I
I
I
I
IS
B
I
IOP
B
K12
K13I K61I N12I N13I N61 H12 H13 H61
Y/// ///A
'////
Y//A,
I
I
I
I
I
IS
I
I
I
IS
I
I
I
I
I
I
I
I
I
I
I
I
I
IO
I
B
I
I
I
I
I
I
V/A Y/A S//A Y//A Y//s///A
OVA, Y///<///A y//A
OY/. Y/A
Y//A V/A
Y//AY/// y/A
i wm
S//AY/A ///A Y/MY///
Y//A Y//A '/A
///A Y/A ///A Y//A Y/As//AY//A Y/// '/A
••i
///A Y/// A/A/t Y//A Y//A,
'/A
,«...
Y/// A//A Y//A< ff///
Y//A <//!//.
Y/A Y/A-.... . -x \-*~-
FIGURE l. Sequence analysis of human melanoma ras genes at codons 12, 13, and 61. PCRamplified DNA from uveal melanoma tumors (No. 1,16, and 29) and cutaneous melanoma
(No. 2C) in the regions of codons 12, 13, and 61 of Ki-2, cHa-rasl, and N-ras are shown dot
blotted on nylon 66 membranes and probed with phosphorus-32-labeled oligonucleotides.
Wild-type panels represent amplified DNA hybridized to wild-type sequences, and mutant
panels represent hybridization with all possible sequences resulting from point mutations at
the codon of interest.
DISCUSSION
Among eukaryotic cells, ras protooncogenes are ubiquitous and putatively subserve a directing function in
cellular growth and maturation.31 Activating point
mutations in these genes have been reported in association with a wide range of human neoplasms including
as many as 5-24% of cutaneous melanomas,23'32 33%
of pulmonary adenocarcinomas,29 50% of colonic car-
cinomas,14-16'30 and greater than 90% of pancreatic adenocarcinomas.13 In view of the association of ras genetic mutations with numerous malignant processes,
we sought to investigate the possibility that ras oncogene activation might also be related to the development of uveal melanoma. We used a selective DNA
amplification technique, searching for ras genetic
point mutations in 53 uveal melanomas. We were unable to amplify ras protooncogenes from ten tumors
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Investigative Ophthalmology & Visual Science, June 1993, Vol. 34, No. 7
2208
#1
#16
#29
#2C
MUTANT WILD-TYPE MUTANT WILD-TYPE MUTANT WILD-TYPE MUTANT WILD-TYPE
Ha 12
Ha13
Ha61
Kl 1 2
• a
P •
D H
13
'61
9 M
12
13
'61
FIGURE 2. Dot-blot analysis of three uveal melanomas (#1, # 16, #29) and one cutaneous
melanoma (#2C) showing point mutations only in the cutaneous lesion.
but could evaluate approximately 75% of the ras genetic domains of interest in the remaining tumors.
Of the uveal melanomas studied, no ras protooncogene mutations were detected. We did observe an
N-ras guanine to adenine point mutation at codon 61
in one of six cutaneous melanomas studied. This finding was consistent in frequency, substitution type, and
location with previously described mutations in cutaneous malignant melanomas.23
It is possible that we missed existing mutations in
the melanomas studied. However, partial direct sequencing of randomly selected ras protooncogenes
from ten melanomas supported the findings of our
screening techniques. In addition, we were able to
demonstrate wild-type sequences for ever)' dot blot
domain amplified. Thus, for mutations to have been
missed, we would have to postulate that the initial tumor DNA isolate contained both wild-type and mutant
sequences and that either the PCR amplification or
the tetramethyl ammonium chloride-washed hybridization was selective for the wild-type over the mutant
sequence. Neither of these hypotheses seems likely, yet
we must be cautious regarding dot blot analysis when
assessing a negative result.
In summary, this study provides no evidence to
support a frequent association between ras protooncogene mutations and premetastatic human mixed cell
uveal melanomas atcodons 12,13, or 61. Although we
suspect it is unlikely, mutant p21 ras gene products
could still play a role in the development of epithelioid
or spindle cell choroidal melanomas or could play a
role in late metastatic disease, neither of which was not
specifically investigated in this study. Activating mutations at other ras protooncogene codons also cannot
be excluded by this investigation.
Key Words
ras, oncogene, uvea, melanoma, mutation
A cknowledgments
The authors thank Drs. T. Dryja and D. Yandell for their
helpful discussions, G. Cowley for his technical advice, and
S. Hairston for her thoughtful contributions. The tissue was
provided through the Collaborative Ocular Melanoma
Center Pathology Center, Daniel M. Albert, MD, Director.
(The Pathology Center is supported by a grant from the National Eye Institute (NEI U01 EY06485).)
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Investigation of ras Protooncogene
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