1 2
of GTPase activity associated with either ras or its
COMPOSITIONS FOR DETECTING RAS GENE oncogenic counterpart remains unknown.
PROTEINS AND CANCER THERAPEUTICS Recently a cytoplasmic factor has been identified
which stimulates normal ras p21 GTPase activity, but
FIELD OF THE INVENTION 5 does not effect GTPase activity associated with the
This invention relates generally to the field of oncol- f^^ZTnL^^ ?k McCormitck|
ogy, and particularly to diagnostic compositions useful 5c'!"ce' 238:542 (1987| The activity has been associated
. °: * A it *t. • with a protein, termed GAP, which is the acronym for
m testing for cancer. Additionally, the invention con- „„„ v '.. . . „AT> . ,„ J ,
"... . , ,. GTPase activatmg protein. GAP is thought to be a
cerns compositions that can be employed both as cancer » • u * • _ ,, r
,. .. v ,i ■ i. c -j • 10 cytoplasmic protein but is presumably capable of mov
diagnostics, as well as in a scheme for identifying cancer • / tV. i. *i. i _ _ _u v.
6 'e ing from the cytosol to the plasma membrane where it
therapeutics. . f . ... r
r mteracts with p21.
BACKGROUND OF THE INVENTION As alluded to above, ras oncogenes have been impli
„ , , . ... cated in the development of a variety of tumors, and
Several genes have been identified that are though to u ^ hem ... to^ mvolved in about 1(M0% of the
play a role in regulating normal cell growth. A subset of mQst ... forms of human ... R y
these genes, termed ras, consists of at least three mem- Annml Rgv Gg lg:553 (19g4) M Barbacid m
bers, N-ras, H-ras, and K-ras2. Altered forms of ras, Important Advances in Oncology (1986), ed. B. DeVita, S.
termed oncogenes, have been imp heated as causative Hd g Rosenbe 3_22> philadelphiaiip
agent in cancer. Both the normal cellular genes, and the 2Q mcoU Fof , ra$ enes have ^ consis.
oncogenes encode chemically related proteins, genen- tendy ... m carcinomas of the bladder) colon>
cally referred to as p21. kidney, liver, lung, ovary, pancreas and stomach. They
Ras oncogenes and their normal cellular counter- ^ have been identified in hematopoietic tumors of
parts, have been cloned and sequenced from a variety of lymphoid and myeloid linea e_ ^ well ^ ^ tumors of
species. Comparison of the structure of these two genes 25 mesenchymai origin. Furthermore, melanomas, terato
has revealed that they differ by point mutations that carcinomaS) neuroblastomas, and gliomas have also
alter the amino acid sequence of the P21 protein. Natu- been shown tQ ras oncogenes.
rally occurring mutations in the ras oncogenes have Considering the possible association of ras oncogenes
been identified in codons 12, 13, 59, and 61. In vitro ^ cancer; there has ^ considerable work focused
mutagenesis work has shown that mutations m codon 30 on diagnostic tests for detecting the presence of the
63,116, and 119 also result in transforming activity. The oncogene product, p21, or the mutant oncogenes. Early
most frequently observed mutation which converts a testS; which ^ still empioyed in many instances, iden
normal cellular ras gene into its oncogenic counterpart tify the presence of ras oncogenes in transfection assays
is a substitution of glycine at position 12 by any other which identify p21 by its ability to transform NIH 3T3
amino acid residue, with the exception of proline. 35 cells Lane> et alf Proc Nath Acad, ScL as A> 1&:5lS5
Transforming activity is also observed if glycine is de- (1981). and B Shil0j mi R A Weinberg, Nature,
leted, or if amino acids are inserted between alanine at 289:607 (1981). This method is insensitive, laborious,
position 11 and glycine at position 12. and requires a skilled laboratory technician to perform
Mutations at position 61 also play an important role in adequately,
the generation of ras oncogenes. Substitution of gluta- 40 A second diagnostic method centers around oligonu
mine for any other amino acid, except proline or glu- cleotide probes to identify single, point mutations in
tamic acid in the cellular ras gene yields ras oncogenes genomic DNA. This technique is based on the observa
with transforming activity. tion that hybrids between oligonucleotides form a per
In relation to normal cellular ras genes and their fect match with genomic sequences, that is, non
oncogenic counterparts, there are at least four known 45 mutated genomic sequences are more stable than those
retroviral ras oncogenes which exhibit transforming that contain a single mismatch. The latter, of course,
activity. Unlike their non-retroviral analogues, the re- being a point mutation in p21 associated with the ras
troviral genes exhibit two mutations. The biologically oncogenes. Although this technique is clearly more
significance of these double mutations is at present un- sensitive and easier to perform than the transfection
clear. 50 assay, it is nevertheless also cumbersome to perform.
Both the normal ras and oncogenic p21 proteins, This is because there are theoretically almost 100 base
regardless of their phylogenetic origin, bind guanine substitutions which can yield ras oncogenes. Thus, in
nucleotides, GTP and GDP, and possess intrinsic order to be able to detect these substitutions multiple
GTPase activity. Temeles et al., Afa/ure, 313:700(1985). oligonucleotide probes must be employed containing
The significance of these biochemical properties to the 55 each of the three possible substitutions at a particular
biological activities of the ras proteins has been demon- residue. Bos, et al., Nature, 315:726 (1985); Valenzuela,
strated as follows: first, microinjection of anti-ras anti- et al., Nuc Acid Res., 14:843 (1986).
bodies that interfere with guanine nucleotide binding In addition to the transfection and oligonucleotide
reverses the malignant phenotype of NIH 3T3 cells assays, additional nucleic acid hybridization techniques
transformed by ras oncogenes. Clark, et al., Proc. Natl. 60 have been developed to identify ras oncogenes. One
Acad. Sci. U.S.A., 82:5280 (1985); Feramisco, et al., such method is based on the unusual electrophoretic
Nature, 314:639 (1985). Second, ras oncogenic proteins migration of DNA heteroduplexes containing single
that exhibit mutations which result in the inability of based mismatches in denaturing gradient gels. Myers et
p21 to bind guanine nucleotides do not transform NIH al., Nature, 313:495 (1985). This technique only detects
3T3 cells. Willumsen, et al., Mol Cell. Biol., 6:2646 65 between about 25-40% of all possible base substitutions,
(1986). Third, some ras oncogenes produce p21 proteins and requires a skilled technician to prepare the denatur
that have much reduced GTPase activity compared to ing gradient gels. More sensitive techniques which are
their normal cellular counterparts. The biological role refinements of this technique are described by Winter,
