28

105

DT-DIAPHORASE AS A DETERMINANT OF SENSITIVITY TO

YlTOYYClN C AND ITS ANALOGUE (KW-2149) IN NON-SMALL

LUNG CANCER CELL LINE UNDER HYPOXIA.

T Bando, K. Kasahara, K. Shibata, Y. Nakatsumi, Y. Numaia, M. Tsujiura.

M. Fulimura. & T. Matsuda. The Third Department of Internal Medicine,

School of Medicine, Kanazawa University. Kanazawa. Japan.

Antitumor activity of mitomycin C (MMC) is enhanced by exposure to

hypoxia. To evaluate the role of DT-diaphorase (DT-D) as a bioactivator of

MMC and its newly developed analogue, KW-2149, in the non-small cell

lung cancer (NSCLC) cell lines under aerobic or hypoxic condition, we

examined the inhibitory effect of dicumarol. an Inhibitor of DT-D, on the

sensitivity to these anticancer agents in vitro In this study, we used MMC-

resistant NSCLC cell line (PC-9/MC4) which was established in our laboratory

from PC9 cell line as a parent cell line by continuous exposure to MMC. The

subline PC-g/MC4 was 6.7-fold more resistant to MMC than PC-g, their

parent cell line, under aerobic condition, and 5.2-fold more resistant under

hypoxic condition. PC-91MC4 cell line did not show collateral resistance to

KW-2149 In PC-9 cells, MMC-sensitivity was significantly Inhibited by

dicumarol under aerobic, but not under hypoxic condition Sensitivity to

KW-2149 was not altered by dicumarol treatment or exposure to hypoxia.

These results support the hypothesis of a partial role of DT-D in bioactivatlon

of MMC, but not of KW2149, under aerobic cundition. and KW-2149 may

be an excellent anticancer agent to circumvent the resistance to MMC.

107

OVEREXPRESSION OF THE MRP GENE IN A MULTIDRUG- RESISTANT SMALL CELL LUNG CANCER SURLINE. LA. Doyle, D.D. Ross, W. Yang, Y. Tong, Y. Gao, c. Belani, and J.C. Gutheil, University of Maryland Cancer Center, Baltimore, MD 21201.

We have characterized an etoposide-resistant subline of the small cell lung cancer cell line UMCC-1, derived from a patient at our center. Suhline UMCC-l/VP was developed by culturing the parent line in increasing concentrations of etoposide over 16 months, and is currently maintained continuously in 4 FM drug. UMCC-l/VP is lOO-fold resistant to etoposide by MlT assays,

‘relative to the parent line, and is cross-resistant to doxorubicin, vincristine, and actinomycin D. Topoisomerase II immunoblotting and functional assays show a 2.fold decrease in the resistant subline. The UMCC-l/VP subline demonstrates a marked decrease in accumulation of 3H-etoposide relative to the parent line as well as decreased daunorubicin uptake by fluorescence microscopy. RT-PCR assays demonstrate no MDRl expression but marked overexpression of the MRP gene in the resistant subline. Northern blotting with a 1 kb cDNA fragment of MRP demonstrates the expected 6.5 kb band in UMCC-l/VP which is not detectectable in the parent UMCC-1 line. 2-D gel electrophoresis of membrane proteins reveals a 190 kD protein in the resistant subline, but not the parent line. MRP gene expression may be induced by etoposide and may lead to reduced accumulation of the drug.

106

CELLMEMBRANBBAND CISPLATINUPTAKEINTOALUNGCANCER CELL LINE AND ITS CISPLATIN-RESISTANT VARIANT. DJ. Stewart, P. Popovic, D. Grewa& R. Gael, M. Molepo, M. K&s, P.T.T. Won& Ottawa Regional Cancer Centre, University of Ottawa, and National Research Counfil, Ottawa,C""ad& WecomparedtheHTB56humanlungadenocarcinomaceUlineaitsHTB56/E-8

(E8)variant (inwhichwehadinduced 3-foldcisplati"resistancebychronifexposure to low dose cisplatin). We previously found by pressure hming infrared specaoscopy (F’TIS) that: a) the OV2008 human ovarian cancer cell line and the EMT6 murk mammary cancer have more fluid cell membranes than their &plain-resistant variants withreducedcisplatinupmke(PAACR33:464,1992;PAACR34:4G5,1993); b)cisplatinappearsabletodiffuseacrossfluidmodelmembranes,bu~notmaerigid ones(PAACR33:448,1992;PAACR 34:401,1993). Itisunlolownwhethercisplatin enters cells by active transport or by passive diffusion.

ES cells had 30% lower &plain UptaLe than HTB56 cells after 1 hour exposure. FTIS showed B 0.6 kbar increase in the disorder-xder transition pressure of methylene chains of "x?tie lipids in ES cells, implying increased membrane rigidity. Compared to HTB56 cells, E8 cells had higher sphingomyeli", phosphatidyletlm"olamine,andcholesterol("llofwhichi"creasemembranerigidity) and lower phospixtidylcholine (the most fluid phospholipid). Cisplatin uptaLei"to HTBMand E8 cells was assessed tithpreincubatio" wco"curre"tincubationwith thetnetabolicinhibitorsdinitrophe"ol,NaF,iodoacetate,fluoroace tate,ouabain,a"d NaN, singly or in pahs. Cisplatin uptake was increased by some,dearased by others,andu"affectedbyothem.. EffectsonHTB.56andE8cellaweresimilarexcqt that ouabain increased cisplatin uptake in E8 cells but decreased uptake in HTB56 cells. Effect of cations (over a physiological range) on cisplatin uptake was tested in HTB56 cells. Cispladn uptake was decreased by N&l, increased by C.&l, and C&l, and not changed by KCI, MgCl, & selenite. Cisplatin efflw. was debased by NaCl and KCl. Hence: 1) Cisplatin-resistant ES cells have deceased cisplatin npcale, increased membrane rigidity, and altered membrane lipids. 2) Metabolic inhibitors and cations alter cisplatin uptake. It is unknown if they alter active transport or if they alter passive diffusion by altering membrane lipid metabolism.

108

IMMUNOHISTOCHEMICAL (IX) STUDY OF TOPOISO- MERASE II (TOP0 II) IN PATIENTS WITH NON-SMALL CELL LUNG CANCER (NSCLC). A. Charm. P. Bourne. L. Boros, Drake F.., DeVdre R., D. jbhnson. The Genesee Hospital,, University of Rochester, Rochester,. NY, SmithKline Pharmaceuticals, King of Prussia, PA, University of Vanderbilt, Nashville, TN. USA.

Topo II is a nuclear enzyme essential in DNA replication and the target for etoposide (E). The higher the topo II content, the more sensitive are the tumor cells to E in vitro. We have developed a microwave IHC method for staining top0 II in paraffin sections. We studied 34 patients with resectable NSCLC and 18 patients with metastatic NSCLC who were treated with E (50 mg/m2/d P.O. x 14 g 3 weeks).. We zqacl;f)the IHC results ?s.negative (no +ini?g

borderline positive (trace staining In less tdan 25% of tumor cells) and oositive staining.

No. of Patients Stainina TlNOMO TZNOMO Tl-ZNl-2M0 Results. Negative 2 2 0 Borderline 6 0 1 Positive 9 6 8

No. of Patients (with Metastatic Disease) Response to E Median Survival (d)

Negative 2SD 3PD 1NE 57 (12-201) Borderline 1PR 2PD 1NE 72 Positive

(57-369) 2PR 4SD 2PD 270 (88-558)

Our results suggest that IHC staining of topo II may be related to stage and aggressive- ness of the disease in resectable NSCLC and an prognostic indicator for advanced NSCLC treated with oral E.