Cancer Letters, 56 (1991)

103-

103

108

Elsevier Scientific Publishers Ireland Ltd.

Effect of protease inhibitors on DNA amplification SV40-transformed Chinese hamster embryo cells

in

M.B. Flick and A.R. Kennedy Department (Received (Accepted

Oncology,

of Radiation

University

of Pennsylvania

Philadelphia,

PA 19104 (U.S.A.)

Introduction

We have examined

the effect

hibitors on radiation-induced

of protease in-

DNA

amplification

in vitro using an SV40-transformed Chinese hamster embryo cell line. DNA from cells irradiated with X-rays (8 J/m21

(10 Gy) or ultraviolet

radia-

in an S-fold or greater amplification of SV40 sequences compared with unirradiated cells. Addition of antipain or the

resulted

soybean-derived Bowman-Birk protease inhibitor (BBI) to the culture medium, either 75

min after X-irradiation or within 15 min after ultraviolet irradiation, resulted in a reduction of amplification levels. BBI was more effective than

antipain at an equal concentration.

These results in DNA

suggest that a protease may be involved amplification.

Keywords: DNA amplification; hibitors; ultraviolet

radiation;

protease X-rays.

in-

Correspondence to: Dr. Ann R. Kennedy, Department of Radiation Oncology, sylvania

of Medicine,

2 October 1990) 19 November 1990)

Summary

tion

School

195 John Morgan Building, University of Penn-

School

of

Medicine,

37th

and

Hamilton

Walk,

Philadelphia, PA 19104-6072, U.S.A. Abbreuiotions: BBI, Bowman-Birk Inhibitor; DHPB, dihydrofolate reductase; EMS, ethyl methanesulfonate; MNNG, nftro-nitrosoguanidine; ultraviolet.

0304-3835/91/$03.50

SDS,

sodium

dodecyl

N-methyl-N’-Nsulfate;

UV,

Normal cell function is achieved through the tightly regulated expression of numerous genes present in all cells. The over-expression of genes can result in significant biological consequences. For example, multidrug resistance has been associated with over-expression of certain membrane protein genes [31]. Over-expression may be the result of transcriptional activation, gene amplification or both. The over-expression of cellular oncogenes is thought to play a role in the initiation and/or the progression stages of carcinogenesis [Z, 111. We have previously demonstrated that protease inhibitors are capable of inhibiting carcinogenesis in vivo and transformation in vitro [Ml. In particular, the soybean-derived BBI is effective in suppressing hamster cheek pouch, liver and oral carcinogenesis in vivo and radiation-induced transformation in vitro. One mechanism by which protease inhibitors suppress carcinogenesis may be through the inhibition of gene amplification. Using a SVLCO-transformed cell line previously reported to undergo selective amplification of SV40 sequences in response to physical and chemical carcinogens [9,13,20,22], we have examined the effect of anticarcinogenic protease inhibitors on UV and X-ray induced gene amplification.

0 1991 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

104

Materialsaadlbthods Chemicals and molecular probes Antipain was obtained from Sigma. The BBI was obtained from Central Soya (Fort Wayne, IN); the methods utilized in its preparation have been described in detail [33]. This BBI preparation is effective at inhibiting transformation in vitro [33], carcinogenesis in vivo [25] and other endpoints related to carcinogenesis [ 171. SV40 DNA (5.2 kb) was purchased from Bethesda Research Laboratories and linearized by digestion with EcoRl . A 1.5-kb mouse DHFR cDNA insert was excised from plasmid pDl1 (the plasmid construct is described in Ref. 24) by digestion with Pstl and isolated by gel electrophoresis. Probes were labeled to greater than 1 x log cpm/pg using a Random Primed DNA Labeling Kit (Boehringer Mannheim Biochemicals). Cell culture Co60 SV40transformed Chinese hamster embryo cells, designated Cl 1, were cultured at 37OC with 5% CO, in Ham’s F12 medium supplemented with 10% fetal bovine serum. Zrradiation

Cells were seeded at 105 per 100 mm plastic tissue culture dish and incubated overnight. Dishes were exposed to an X-ray source (Phillips 100 kVp Irradiator, 2 mm aluminum filter, dose rate of 1.8 Gy/min) to accumulate 10 Gy. Survival under these conditions was 8%) as determined by a colony forming assay. For UV irradiation, the medium was removed and the dishes were placed uncovered under UV light for a length of time to accumulate a dose of 8 J/m2 (2% survival). Cells were irradiated with a single UV lamp (American Ultraviolet Co. 782L30) calibrated with a YSI radiometer. DNA extraction and slot blot At defined time intervals, cells were trypsinized and counted. Sample volumes equivalent to 5 x lo4 cells were centrifuged (12 000 x g, 5 min, 4OC) in 1.5-ml microtubes to pellet the cells. The medium was removed and the pellets were stored at -8OOC until use. DNA was ex-

tracted by a modification of the method of Brown et al. [5]. To frozen cell pellets, 10 ~1 of 10 mM Tris (pH 8.0), 10 mM EDTA, 10 mM NaCl (TE,oN) containing 0.2% SDS was added and incubated for 10 min at 37OC with vigorous shaking. Samples were diluted with 180 ~1 TE,,N and 8 ~1 of ribonuclease A (0.5 mg/ml) was added, followed by incubation at 37OC for 1 h. Proteinase K (5 mg/ml) was added to a final concentration of 50 pg/ml and the samples were incubated for 1 h at 37OC. NaCl was added to a final concentration of 0.1 M and the mixture was extracted twice with equal volumes of phenol/chloroform (1: 1, saturated with TE,,N) and chloroform. The aqueous phase was transferred to fresh tubes, 20 ~1 of 2 M NaOH plus 0.2 mM EDTA was added and the samples were incubated for 1 h at 65OC. The samples were neutralized by the addition of 20 ~1 of 3 M sodium acetate, diluted by adding 600 PI of 20 x SSPE (1 x SSPE = 0.15 M NaCl, 0.01 M NaH2P04, 0.001 M EDTA; pH 7.4) and applied to a slot blot apparatus (HybriSlotTM Manifold, Bethesda Research Laboratories) using nitrocellulose or nylon (Nytran, Schleicher and Schuell) membranes backed by two sheets of GB002 filter paper (Schleicher and Schuell) all prewet with 15 x SSPE. Membrane filters were baked for 30 min in vacua at 8OOC. Hybridization

and autoradiography

Filters were prehybridized in 5 x Denhardt’s (1 x Denhardt’s solution = 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone and 0.02% Ficoll400), 5 X SSPE, 0.2% SDS, 0.5 mg/ml denatured salmon sperm DNA at 65OC for 2-4 h and hybridized with 32Plabeled linearized SV40 DNA at 65OC for 20 h. After washing (2 x 15 min at 25OC with 2 x SSPE, 0.2% SDS and 2 x 65OC in 0.5 x SSPE, 0.2% SDS), the filters were exposed to Kodak XAR film for 18-72 h. Hybridization signals were quantitated by densitometric scanning of the autoradiograms using a LKB UltraScan laser densitometer. Areas under the curves were used to compare the levels of hybridization in treated vs. untreated cells. Prior to reprobing, the nylon filters were strip-

105

chemical carcinogens, including tumor promotors [1,9,13,19,20,22,27,29,32]. In addition, amplified oncogenes have been found in a large number of tumorigenic cell lines and tumor types, including primary human osteosarcoma cell lines [3], human ovarian carcinomas [lo], a human melanoma cell line [ 121, primary rat tumors [26] and human breast cancer [28]. DNA amplification has also been correlated with tumorigenicity, metastatic potential and disease prognosis [8,12,28,30] and may confer a selective advantage on tumor cells [8]. Therefore, while gene amplification is considered by some to be a secondary event in tumor progression [8], its inhibition may retard or block the development of malignant cells. Treatment of cells with antipain or the BBI at 40 pg/ml did not result in significant changes in copy number of SV40 DNA sequences in unirradiated cells after 3 days in culture (Tables I and II), and further, did not have any toxic effects on cell growth. Exposure of cells to a UV dose of 8 J/m* produced a 14-fold and 8-fold amplification of SV40 DNA sequences after 2 and 3 days incubation, respectively (Table I) _

ped of previous radioactive probe according to the manufacturers instructions. To determine the relative amount of DNA applied per slot, the filters were reprobed with the non-amplified DHFR gene [ZO]. Filters were hybridized at 58OC for 42 h and washed (2 x 15 min, 25OC and 2 x 15 min, 58OC in 2 x SSPE, 0.2% SDS). No significant difference in DHFR hybridization signals was observed between unirradiated and irradiated cells or between irradiated treatment groups indicating that the same amount of DNA was applied to each slot. In control experiments, known amounts of Cl1 cell DNA (0.14-36.2 fig) from control or UV-irradiated cells were slot blotted and hybridized to [32P]SV40 DNA. The autoradiograms were scanned and the hybridization signals plotted as a function of the amount of DNA applied. The film response was linear with the amount of DNA applied to the filters (not shown).

Results and Discussion Gene amplification is one of several responses of mammalian cells to both physical and

Table 1.

Effect

of protease inhibitorson ultraviolet-induced Treatment

Group

amplification

of SV40 DNA sequences

SV40 hybridization

None Antipain only BBI only UV only UV + antipain UV + BBI

3

4 5 6

cells.

post-treatmenta 3 days

2 days :

in transformed

0.082 0.057

f

0.092 0.815 0.874 0.605

f f f f

0.056 0.014 0.012 0.118 0.128 0.147

0.092 0.104

f

0.013 0.020

0.102 0.854 0.673 0.650

f 0.011 + 0.107 f 0.062 * 0.131

t-test analysis of data Comparison

1 vs. 1 vs. 1 vs. 4vs. 4 vs.

2 3 4 5 6

P-value 2 days

3 days

0.161 0.320