26

091

EFFFCl-OFlWCXSOMERASEIILJ?VEUONCOMPARATlW

CHEMOSENSITIVlTY OF LUNG CANCER CELL LINES. J. Cannichaell,

S. Houlbrookl. I. Kirkl. N. St&. A. Fry2. A. Harrisl. ‘ICRF Clinical

Oncology Unit. Churchill Hospital. 21nstitute of Molecular Medicine, Oxford. The chemosensitivity of a panel of lung cancer cell lies was determined to a

range of drugs including 3 drugs (m-AMSA. VP16. adriamyein) known to have their effect. at least partially, through the action of topoisomerase U. Cytotoxicity was compared by measuring the IC50 using the M’lT assay.

The accompanying table shows the IC50 al v “es of the cell lines to 4 of the

drugs examined, listed in order of their sensitivity to AMSA. There is a correlation

in rank sensitivitv to AMSA. VP16 and adriamycin but, as expected less

60.2~12.0

NCXH460 72.5kl2.9 NCI-N417 84~19

A54 103.4*71.3

NCI-H841 238.4*146 NCI-H226 L 470.4k158.8 NC&H522 481j+i

NCI-H526 6192251

NCI-H322 1038661

NC&H358 1582+670 7

lopolsomerase n levels we

I.

i Te d

2.720.8 52.7+1.2 21.8k3.8

1.7kl.2 28.1+12.5 8.122.9

l.lL1.2 29~3 1 1q11

5.7d.9 96.901.9 51.8k39.6

7.921.2 lSgt76.9 19.8rtlO.l

9.8i2.3 275.lk29.5 145.hl.O

10.923.9 45.0+_5.3 19.3k5.4

0.49k.03 122*20 ND

64.7213.0 423.6k79.8 238k79.2

19.9+6.8 1 298.6+103 175+14.4 - . . . . 1etemuneo by western oiottmg o* nuclear extracts 0

exponentially growing cells which were elecaophoresed using 7.5% SDS-PAGE. After transblotting. topoisomerase II was determined by binding of specific

antibodies raised to peptides of topoisomerase 11 a and b sequences followed by

1251 labelled protein A. Varying levels of topoisomerase 11 were found in the nuclear extracts with very low levels observed in 2 cell limes. One cell line (NCI- H460) was. surprisingly, very sensitive to all the drugs tested despite low levels of topoisomerase 11 suggesting an alternative mechanism of resistance. This is currently under investigation. Two cell lines were relatively resistant despite high levels of topoisomerase II. and enzymatic activity is being assessed using a decatanation assay. Correlation between chemosensitivity and topoisomerase II levels was observed. but these results suggest that alternative mechanisms are also of importance in the drug resistance observed in lung cancer.

093

EFFECT OF IL-2, IL-6, AND IL-8 TRANSFECTION ON THE REJECTION OF LEWIS LUNG CARCINOMA Y.Ohe, E.R.Podack, K.O.Podack, Y.Shirahige,

YMiyahara, T.Tamura, K.Miura, S.Kubo, T.Morikage and N.Saijo. Department of Internal Medicine, National Cancer Center Hospital and Pharmacology Division, National Cancer Center Research Institute, Tokyo 104 Japan. Department of Microbiology and Immunology, University of Miami School of Medicine, Florida USA.

In order to develop the mote effective method of immunotherapy we have transfected IL-2, IL-6 and IL-8 gene into Lewis Lung Carcinoma (LLC) cell. mIL-2, Hun-6 and mIL-8 genes have been introduced into neomycin resistant gene containing expression vector, BMGNeo and they were transfected into LLC. LLC transfected with cytokine gene produced the specific cytokine into the culture supematant. When 1 x 106 of UC-IL2 cells were transplanted into C57BL/6 mice subcutaneously all the mice rejected them. Survival times of LLC, LLC-Neo, LLC-IL6 and LLC-IL8 transplanted mice were 42.1k7.9, 34.3k7.1, 17.M3.1 and 33.6rb6.4 days (mean+SD, n=6), respectively. Survival time of LLC-IL6 transplanted mice was significantly shorter than that of LLC transplanted mice (pa.001) without difference of tumor growth. Treatment of rhlL-2, 1 x 10sU / 5 times per week for 3 weeks could not prolong the survival of LLC transplanted mice. Not only nude but also C57BU6 beige mice could not rejected LLC-lL2. However, survival time of LLC-IL2 transplanted nude and beige mice were longer than that of LLC transplanted normal mice. These data suggested that IL-2 gene transfection could effectively induce the immunity against LLC although standard immunization could not.

092

Gene Analysis of puloonary

Lymphoproliferative disorders

Tetsuhiro Shiota”. lataru Chlba”, Sadao Ikeda3’. Nobuhiro Ikei”

Otora Hospital I’. Kyoto Katsura Aospital Respiratory Center”.

Otsuka Assay Lahoratories3’

le performed gene analysis of pulmonary Iynphoprollferatlve

disorders. Cell suspensions were ohtalned from tissues of malignant

lymphooa or pseudolymphoo in Cases 1 to 4. Aigh~oolecular~reight

DNA aas extracted from these specimens, digested with restrlction

endonucleases. size-fractionated by asarose-gel electrophoresis and

transferred by the Southern procedure to nitrocellulose. Bibridiz-

atian to nick-translated “P DNA probes of the inounoglobulin JH,

c,. Cl. regions. and T cell receptor B 1 region. In case 1 and 2.

which were diagnosed B cell lyophoma. cells from both tumors had re-

arranged heavy chain. genes. clerarly established the clonal nature.

In Case 3. and 4 rhlch sere deagnosed as pseudolymphooa. the tuaor

contalned clonal imnunoglobulin gene rearrangenents as detected with

both the .l~ heavy chain and Cn light chain gene probes.

le conclude that gene analysis is an effective procedure for estab-

lishlng a diagnosis of lymphoma in neoplastic disorders of uncertein

cell type and for detecting clonal T cell or B cell populations rlth

atyplcal lymphofollicular hyperplasia.

094

Fxpressim of the nultidrug resistfrce related n&-l gene in hum ltmgarxl~ ltmg cancer

J. lorem’, T. Frie&ergz, M. Mitze3 W. F&h’ R. klinz’ l=3rdkdical Departm%h, 2 = Ins&u& of Toxhlqy, 3 = Depart- nrmt of Oynecolcgy & Cbstetrics, lbiversity of kbi.nz, &nmny

Cverexpressim of the ark-1 gene in caccer cells is associated with pleiotrqic resisrence to cytotoxic drugs often used in ltmg cancer ~andrrdr-lisexpressedinti~rmfrcntedtoahigfi- d%,ofxenobiotics.hbmaasmcdthe~exptessi~inpllrrcnary tissues and 0nors using three diffetwt tmthxls: Rl4 hybridisati~ witllthen&ases1motectiQlassayzdirml_&lotintissic~ rrogenates as *I1 as imnmohisto&raist.ry in biopsies fron previmly umreamd lung cancers tissuespscimmsfmn38tmleatd5fomlepatimts. A 1,5-4 fold overexpressicn of nrlr-lm t+as tmasumd in 9/42 tu- nors. ‘lhs expreasicn m > 4 fold in one &no Lc. 3/16 adeno, 3/16 qumrus cell, O/2 smll cell, tut 3/6 large cell Lc and l/3 carei- raid tumrs slxx& overexpressicn.?hererzasnorelaticntototu- DU~gE&OFSti3gCPIKJt&l ~icnint4morlmogmateswas rrore prevalah (14/43 tlnurs). Immnchistobemical staining of Ql- nor cells lrBs obser& in cnly toe case, bit infiltrating tmcro- Ihageswzreirmrnocreativeingeneral.Lungtissue~r0over- expressicn in all bX@nams. Brond7ial, alveolar and imerstitial ~llcqxnmts~enegativein~stry,b.rtagain~ $lagesIIRI-e imNlomXXt.ive.

Ccozlusico: wlmxlary tiwres and derived cancersQnotf~ @Y exw=5 mh-1: Znq m tilw to OVA b-1 rt+

~atedpleiatrcpicreslstencemayncrtteadiveti~Lc.?he expE5simOflnllti~~inalveolar~mry

have a role in the -is of xcrcbiotics.