Environmental and Molecular Mutagenesis - Legacy Tobacco ...

80 1989 EMS Abstracts 227
http://legacy.library.ucsf.edu/tid/clb93d00/pdf
Notes AN OPTIMIZED PROCEDURE FOR CULTURING RAT LYMPHOCYTES FOR IN VITRO CYTOGENETIC
SCREENING . P.J. G azie and Y. OaWro, O.D. SearM & Co .. Skokle, IL .
Treatment prooedures and culture conditbns were optimized for the tn vitro treatment of peripheral blood
lymphocytes (PBLs) of rats for oyto0enstb evaluation . Approxktttlaly 12-16m1 of blood was aolMded by cardisc
puncture Into heparinl2ed VACUTAOJERTN tubes . Whole blood cultures and FboR4epaated ytnphooyla wan
cuUured at 37'C for 48 h In RPMI-1824 medium supplemented with 20%fetal bovine sennn, L-pkAamMn (2 mM) .
peniciuiNstreptomydn and the mitogen, ConcanavaNrt A(Con A : 6.7 Npht* . Various a>aure aondtbns were
tested Including the addition of agents whidt have been reported to Increase blaslopenaN of ymphooytes .
The addition of UCI (0.3 mM) to cultine medium aotltatNtq Con A did not slpNfioarMy Yanaa the ntlbfb Index
relative to the use of Con A alone . However, the addition of 2-mathoxyelharal (2-ME ; 0 .9 µM) to medium
contalninp Con A produced an 8-told karease In the mitotio Index compared to that observed with Con A alotw .
M approxhnate 6-fold krorase tn numbent of mltotio oaNS was observed with FbDDN-teparaled cultures compared
to whole blood cultures oodaininp both 2•ME and Con A . The effect of c.u density on tM mltadc kdex was
studied by culturing Flooll-separated PBL's at densltNs of I X 108, 1 .6 X 106, and 2 X 106 oa6s per culture .
Maximum mitotic Indices (6-7%) were achieved when 1 .6 X 108 PBLS were cultured In RPMI medium
supplemented with Con A, and 2-ME for 48 h . In addition, cell cycle kinatkn were defined using
5-bromodeoxyuridine In the culture medium to dineretttlale tha propottbtr of cells that had undarpona 1, 2 or 3
cen .divisions over a defined period of 6me . TheN sabNs Indicated an avlrape oeM division tlme of approxknatey
14-16 h after an InRial delay of 24-28 h . TrlelhylensrnslatNne (TEM ; 0.06 mphnq, a dUect-actUq daslopen, was
added to the culture medium for 4 h after a 24 h cuMura period . Lymphocytes were harvested after a total of 48 h
(final 3 h with coloemid) In culture and evaluated for chromoaome damage . Approximately 37% of the
TEM-treated cells were aberrant compared to oottirol levels of 3-4% . Additional known dastopans and
non-clastogens are being tested to evaluate thk test aystem for roupne aonankp of potential dastooens .
POINT ttUTATIOMS IM THE K-RAS ONCOGRa,tt IY DMA-SAHPLSS FROIt LUNG CANCER PATIENTS
228
Hackman, P ., Husaafvel-Pursiainen, K ., Mttila, S ., KalliomYki, P .-L ., HeikkilY, L .,
Hattila, S . & Vainio, H ., Institute of Occupational Health, Topeliuksenkatu 41 aA,
00250 Helsinki, Finland
Activation of the cellular onco6enes in the rn-Sene family has been implicated
in many types of human piali6nancies . In this work we are studyina the mutational
activation of K_ras-onco6enes in lunS cancer patients . Peroperational lung tissues
as well as blood samples from 21 lunb cancer patients, were obtained from the
Helsinki Universlty Central Hospital . DnA was isolated from tumour tissue as well as
from normal peripheral lung tissue, and from wfiite blood cells and stored freeze
dri•ed . All of these patients were smokers or ex smokers . Of the 21 lung cancers
twelve were squamous cell carcinomas, six were adenocarcinomas and three small cell
carcinomas . Fourteen of these patients had a history of occupational exposure to
asbestos . DNA-sequences from samples of human DNA were amplified using the
PCR-technique (Polymerase Chain Reaction) . The amplimers used in this reaction were
identical with sequences situated on both sides of the human K-ras-oncoSene exon 1 .
Codons 12 and 13 which are located in this exon have been reported to be amon6 the
critical points for the mutational activation of the K_ras-onco6ene . Sxperiments are
in proaress to analyse the amplified DHA for specific point mutations by
hybridization using specific 20 bp oli`onucleotldes . Wild-type oli6onucleotides as
well as at least 6 "mutated" oli6onucleotides differinS in one base pair/codon are
used in these experiments .
229
THE ORIGIN OF MIlTA[JT3 . Bany 0 . Hall . Dept. of Biology, University of Rochester, Rochester, NY .
Spontaneous mutations are assumed to occur randomly, condntxxtsly, and without regard to their utility .
Two studies from my labaatory suggest that some mutations occur more often when they are advantageous
than they do when they are irrelevant to the well being of the cell . In both cases these spontaneous ad hoc
mutations occur in aged, nutritionally depleted, colonies of BschertchJa coll . In the first study excision of a
mobile DNA element, IS103, from within the bglF gene is required for expression of that gene which then
permits utilization of the 8-glucoside sugar salicin . The excision event is undetectable (Q x 10-12 per cell
division) in cultures of growing cells, but it occurs at frequencies as high as 10'1 in aged colonies on
MacConkey plates if, and only if, salicin is present in the plate . In the second study reversion of point
mutations within the trp operon of E . coli occur 15 to 25 fold morefrequently In aged colonies on
tryptophan depleted medium than they do in growing cells . A variety of trivial explanations for these
observations are ruled out, and it is concluded that the vast bulk of the mutants recovered in these
experiments are the result of mutations that would not have occurred under non-selective conditions by
chance alone. It now seems likely that spontaneous mutation rates are not inherent properties of organisau,
but instead that they are subject to modulation by normally encountered envtronmental conditions .
Furthermore, the probability of a particular mutation arising may depend strongly upon the selective
advantage conferred by the mutation.
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