myc Family DNA amplification in small cell lung cancer cell lines and tumors

Lung Cance~ 4 (1988) 131-134

131

Elsevier mye FAMILY DNA AMPLIFICATION

IN SMALL CELL LUNG CANCER CELL LINES AND TUMORS

BRUCE E. JOHNSON AND ALFREDA SIMMONS NCI-Navy Medical Oncology Branch, Building 8, Room 5101,

Naval Hospital,

Bethesda Maryland 20814 USA INTRODUCTION myc family

DNA amplification

and mRNA expression has been shown to play an important

in the biology of small cell lung cancer, associated

and mRNA expression has been

with a variant subset of small cell lung cancer cell lines which have more aggressive

biologic characteristics proto-oncogene

than the more common classic phenotype

(1-4).

Transfectlon of the c-myc

leading to expression of c-myc mRNA in a small cell lung cancer cell llne which

did not express addition,

c-myc DNA amplification

role

c-myc mRNA reproduces

c-myc DNA amplification

some of these aggressive growth characteristics

(5).

In

has been associated with shortened survival in treated small

cell lung cancer patients who have had tumor cell lines established

(6).

other myc family members,

in small cell lung cancer

cell lines and tumors studied

N-myc and L-myc,

(7-i 2).

have also been described

The incidence of m vc family DNA amplification

in paraffin embedded small cell lung cancer tissue (13).

amplification described.

in unprocessed

However,

of

has also been

the incidence of DNA

cancer tissue from small cell lung cancer patients has not been

We therefore decided to systematically

tumors from small cell lung cancer patients amplification

DNA amplification

evaluate

the DNA prepared from cell lines and

treated at our branch.

pattern was correlated with the clinical presentation

The myc family DNA and outcome of the small cell

lung cancer patients and the results are reported here.

METHODS Cell lines were grown in RPMI 1640 plus 10% fetal calf serum and were harvested in log phase growth.

Tumors were obtained

examinations.

to nitrocellulose

l-Eco RI 3rd exon fragment

(14).

The DNA was digested with restriction endonucleases,

paper (15) and hybridized

to radiolabelled

c-myc probe, a Cla

(16), N-myc, an Ec___~oRI-Ba___mmHI second exon fragment

Sma l-Eco R1 third exon fragment (7). prepared.

and 30 post-mortem

The tumors were finely minced and the DNA was isolated from the cell ines and

tumors by the method of Heiter transferred

from 4 surgical biopsy specimens

The DNA was considered

(17), and L-myc,

to be amplified if the signal intensity was found to be four

fold greater than a single copy control.

The clinical course of the patient was reviewed

and survival duration from the initiation

of therapy was noted.

establishment

a

The blots were exposed to x-ray film and autoradiographs

of the cell line or procurement

In addition,

the date of the

of tumor tissue was noted.

RESULTS myc family DNA amplification

in small cell lun~ cancer cell lines

The DNA from cell lines established f or amplification

of

c-myc, N-myc and L-my c.

cell lines had c-myc amplification, (Table 1).

The DNA amplification

chemotherapy

untreated

The DNA from 7 small cell lung patients'

5 had N-m~c amplification,

small cell lung cancer patients.

from chemotherapy

tumor

and 6 had L-mycampllfication

was more common in tumor cell lines established

treated than untreated

cell lines established family genes

from 66 small cell lung cancer patients was examined

from

Fifteen of 35 (43%)

treated patients had-DNA amplification

of one of the myc

(7 c-myc, 4 N-myc, and 4 L-m~c) compared to 3 of 31 (10%) cell lines established

patients (I N-myc and 2 L-myc) .

Chemotherapy

whose tumor cell lines had c-mzc DNA amplification of chemotherapy

from

treated small cell lung cancer patients

lived a shorter period of time from initiation

than similar patients whose tumor cell lines did not have c-myc DNA amplification.

0169-5002/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

132 TABLE I. mye FAMILY

DNA AMPLIFICATION

CHEMOTHERAPY

OF SMALL CELL LUNG CANCER CELL LINES AND ITS RELATIONSHIP

TO

TREATMENT

Chemotherapy

Treated

Untreated

c'-m.yc

7/35

N-myc

4/35

1/31

L-myc

4/35

2/31

TOTAL

15/35

3/31

The chemotherapy amplification

0/31

treated small cell lung cancer patients whose tumor cell lines had c-myc DNA

lived a median of 27 weeks

(range 13-58) compared

to 49 (range 19-112) weeks in

patients whose tumor cell lines did not show c-myc DNA amplification. myc family DNA amplification The DNA amplification was also studied

in small cell lun~ cancer tumors

pattern in tumors taken directly from small cell lung cancer patients

(Table 2).

The DNA from six patients who had received chemotherapy

having the tumor harvested was amplified None of the tumors obtained had DNA amplification

TABLE

prior to

for one of the myc family genes (4 N-myc and 2 L-myc).

from six patients who had not been treated with chemotherapy

of any of the m~,c family genes.

2.

myc FAMILY DNA AMPLIFICATION CHEMOTHERAPY

OF SMALL CELL LUNG CANCER TUMORS AND ITS RELATIONSHIP

TREATMENT

Chemotherapy

Treated

Untreated

c-m_,y_£.

0/28

0/6

N-myc

4/28

0/6

L~_~

2/28

0/6

TOTAL

6/28

0/6

The incidence of myc family DNA amplification established

TO

from patients

treated with chemotherapy

was more common in the tumor cell lines (15/35; 43%) studied here than in the tumors

obtained directly from similarly treated patients (6/28%; achieve statistical

significance

finding of myc family was unusual.

21%) although this difference did not

at the p~0.05 level using a chi-square

DNA amplification

analysis.

The

in tumors or tumor cell lines from untreated patients

Only 3 of 37 (8%) of untreated patients studied had DNA amplification

of one of the

myc family genes.

DISCUSSION myc family DNA amplification tumor tissue

of small cell lung cancer cell lines (I-4, 6-12), unprocessed

(7,9) and paraffin embedded

The percentage of myc family

tissue

DNA amplification

(13) has been reported by numerous investigators. in small cell lung cancer cell lines reported here

(20/66, 30%), is within the range of other recent reports of 2/4 (8), I/I (I0), 0/6 (II), and 5/6 (12) from studies of smaller numbers of cell lines. amplification

Although examples of myc family DNA

in tumors obtained directly from small cell lung cancer have been reported

this study reports the incidence of myc family patients whose tumors are not amplified.

DNA amplification

The incidence of myc family DNA amplification

cell lung cancer tumors reported here (6/34,

18%),

(7,9),

in tumors by also reporting the in small

is similar to that reported by Wong (13) in

133 their study of DNA extracted

from paraffin embedded small cell lung cancer tissue (5/45,

The results here extend our previous studies in small cell lung cancer. reported an association chemotherapy established

betweeen myc family

We have extended our analysis i0 established

from 44 tumor cell lines to 66.

chemotherapy

that myc family DNA amplification

and 12 from untreated patients,

family genes.

from the chemotherapy

In contrast,

treated patients was amplified for one of the myc

DNA from only 3/31 (10%) of the cell lines established

in cell lines established

to that reported previously exclusively

(5).

in tumor specimens

only 6 untreated patients'

This difference

between myc

from treated and untreated patients is similar

The study of tumors showed myc family DNA amplification

from chemotherapy

treated small cell lung cancer patients although

in tumor cell lines established

from chemotherapy

small cell lung cancer patients has been associated with shortened survival extended our previous observations after chemotherapy

on 25 patients

treatment.

lines had c-myc DNA amplification of 49 weeks

from untreated

tumors were studied.

c-myc DNA amplification

established

from

DNA prepared from 15 of 35, 43%, tumor

small cell lung cancer patients had myc family DNA amplification. DNA amplification

the previous

was more common in cell lines established

treated than untreated patients was still true. cell line established

With the addition

from tumors obtained from small cell lung cancer patients who

had been treated with combination

family

We have previously

in tumor cell lines and and previous

treatment of the small cell lung cancer patients from whom these cell lines were (6).

of 22 cell lines,

observation

DNA amplifcation

11%).

treated

(5).

We have

to 35 patients who have had cell lines

The 7 chemotherapy

treated patients whose tumor cell

lived a median of 27 weeks (range 13-58) compared to a median

(range 19-112) for patients whose tumor cell lines were not amplified for c-m~c.

In addition to this clinical association of c-myc DNA amplification cell lines with shortend patient survival, of c-myc DNA amplification

Gazdar and Carney have also reported an association

with a variant phenotype of small cell lung cancer cell line which

has more aggressive biologic characteristics have been reproduced by transfecting does not express proto-oncogene

in small cell lung cancer

c-myc mRNA (5).

plays an important

(1,2).

Some of these aggressive characteristics

a c-myc gene into a small cell lung cancer cell line which

This evidence suggests

that amplification

of the c-myc

role in the biology of small cell lung cancer.

study of myc family DNA amplification

will hopefully continue

The continued

to provide insights into the

biology of small cell lung cancer.

ACKNOWLEDGEMENTS The opionions

or assertions

contained herein are the private views of the authors and are not to

be construed as official or as reflecting

the views of the Department of the Navy or the

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2.

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