Detection of DNA Alterations in Human Bladder Tumors by DNA Fingerprint Analyses Eva Agurell, Ronggui Li, Ulf Rannug, Ulf Norming, Bernhard Tribukait, and Claes Ramel
DNA fingerprint analyses were used to examine the constitutional and tumor DNA from 22 bladder tumor patients. DNA alterations, such as loss of bands, new bands, and intensity shifts were observed in 10 of the 22 patients. The most frequent DNA alteration, occurring in 80% of the patients, was a complete loss of one or several bands. Fingerprint abnormalities were present both in lowmalignant superficial tumors and in high-malignant invasive tumors, but were also lacking in the latter group. Apparently no relationship exists between fingerprint abnormalities and gross chromosomal aberrations or the proportion of S-phase cells as measured by flow cytometry or development of recurrent tumors during a limited observation period. Thus, whether fingerprint aberrations express genetic alterations directly involved in the malignancy potential of bladder carcinoma remains an open question.
ABSTRACT:
INTRODUCTION
Development of tumors is a multistep process, which usually implies a series of mutational changes that can include almost all conceivable mutational events [1]. In mutation research of cancer induction and in genetic toxicology in general, emphasis has been placed on conventional endpoints, i.e., point mutations and chromosomal aberration, whereas other changes, such as gene amplification, genetic recombination, and insertion mutations have received far less attention [2]. Highly repetitive DNA sequences are of interest from this viewpoint. Alterations of such a system should include endpoints other than point mutations and chromosomal aberrations, in particular gene amplification and somatic recombination. An analysis of such alterations during tumorigenicity may help shed light on the mutational process during cancer development. Hypervariable minisatellite DNA constitutes a system suitable for investigations of such alterations. These highly repetitive DNA sequences are dispersed throughout the human genome in different polymorphic loci. The polymorphism at these loci are the result of variations of the number of tandem repeats of a short common core se-
From the Department of Genetic and Cellular Toxicology, Wallenberg Laboratory (E. A., C. R.); Department of Genetics (R. L., U. R.), Stockholm University; Department of Urology, South Hospital (U. N.); and Department of Medical Radiobiology, Karolinska Institute (B. T.), Stockholm, Sweden. Address reprint requests to Eva Agurell, Department of Genetic and Cellular Toxicology, Wallenberg Laboratory, Stockholm University, S-106 91 Stockholm, Sweden. Received August 19, 1991; accepted January 27, 1992. © 1992 Elsevier Science Publishing Co., Inc. 655 Avenueof the Americas, New York,NY 10010
quence. These hypervariable minisateUite sequences have been extensively studied and characterized by Jeffreys et al. [3, 4]. The techniques used are Southern analyses of restriction enzyme-treated human DNA after gel electrophoresis in combination with 32p-labeled minisatellite probes. The two multilocus probes 33.15 and 33.6, with a similar core sequence, have been used to produce individual specific DNA fingerprints in humans and animals [5]. Several human single-locus probes were also developed by Jeffreys et al. [6] to study spontaneous mutation rates at individual hypervariable loci and to determine a l l e l e frequencies in different populations. DNA fingerprint analyses have been used to establish sibships in human pedigrees [7], to clarify paternity disputes [4], in forensic work [8], and in analyses of animal populations [9, 10]. Recently, several investigators examined a variety of tumors to study DNA alterations in tumor tissues with the DNA fingerprint technique [11-14]. They noted altered DNA fingerprint patterns in 30-85% of the tumors. We describe the results of 22 tumors of the bladder studied by DNA fingerprint analysis. Cell morphologic and ploidy characterization of the tumors was also performed. The relation between the altered DNA fingerprint patterns and the result of the cell morphologic and ploidy examination are discussed. MATERIALS AND METHODS Patients and Tumors
Tissues from 22 consecutive patients (mean age of 65 years) referred for tumors of the bladder were obtained by transurethal resection. Tumor stage and grade were evaluated ac53 Cancer Genet Cytogenet60:53-60 (1992) 0165-4608/92/$05.0O
54
cording to the criteria of the UICC and the W o r l d Health Organization (WHO) [16]. Eighteen of the tumors were transitional cell carcinomas, one was a squamous cell carcinoma, and one was an adenocarcinoma. One was a T4 a d e n o c a r c i n o m a of the prostate, and one was identified as a malignant l y m p h o m a . Part of the material in saline was p r e p a r e d for DNA flow cytometry as described in detail p r e v i o u s l y [17]. The tumors were s u b d i v i d e d according to their p l o i d y into d i p l o i d (2c) and a n e u p l o i d of various degree. In addition, the p r o p o r t i o n of cells in S-phase was evaluated.
Preparation of DNA Blood. DNA from leukocytes of the h e p a r i n i z e d blood was p r e p a r e d according to the m e t h o d of Jeffreys et al. [4]. Tumor tissue. T u m o r DNA was prepared according to the m e t h o d of Sambrook et al. [18] with m i n o r modifications. Bladder tumors were kept at - 2 0 ° C until the DNA was prepared. The solid tumor tissue was m i n c e d and susp e n d e d in buffer A (10 mM Tris-HC1 pH 8.0, 10 mM EDTA pH 8.0, and 10 mM NaC1; E. Merck, Darmstadt, Germany). The tumor s u s p e n s i o n was w a s h e d once with buffer A and was then r e s u s p e n d e d in buffer A. S o d i u m dodecyl sulphate (SDS, Sigma Chemical, St. Louis, MO) was a d d e d to a final concentration of 0.5%, and proteinase K (Sigma) was a d d e d to a final concentration of 100/~g/ ml. The mixture was incubated for 16 hours at 37°C with shaking; the mixture was then extracted with p h e n o l : chloroform : i s o a m y l a l c o h o l (PCI) 24 : 24 : 1. RNase A (Sigma) was a d d e d to a final concentration of 40 ~g/ml to the water phase, and the mixture was incubated at 37°C with shaking; after 1 hour, SDS (final concentration 0.5%) and proteinase K (final concentration 100 /~g/ml) were a d d e d to the tube. After 1-hour incubation at 37°C with shaking, the mixture was extracted twice with PCI and once with chloroform. The DNA content in the samples was d e t e r m i n e d with an ultraviolet (UV) s p e c t r o p h o t o m e t e r by reading the absorbance at 260 nm. The purity of the samples was checked by the ratio of the absorbance at 260 n m and 280 nm.
DNA Analysis DNA from both normal and t u m o r tissue of each patient was digested with restriction enzymes HinfI or HaeIII (Betheseda Research Laboratories, BRL, Gaithersburg, MD) according to the m e t h o d of Jeffreys et al. [4]. Ten micrograms DNA was digested for 16 hours at 37°C with HinfI or HaeIlI with a d d i t i o n of 4 m M s p e r m i d i n e (United States Biochemical, Cleveland, OH). The s p e r m i d i n e was a d d e d to aid in the cutting of DNA. DNA was extracted with PCI and chloroform and recovered by ethanol precipitation. The resulting DNA fragments of each patient, normal and t u m o r DNA, were run side by side in a 20-cm-long 0.8% agarose (Bethesda Research Laboratories) gel in 1 x TBE (89 mM trisborate, 89 m M boric acid, 2 mM EDTA) for 24 hours at 50 V at room temperature. The separated DNA fragments were transferred according to the method of Southern [19] in 10 x SSC) 1.5 M NaC1, 0.15 M trisodium
E. Agurell et al. citrate) to a Hybond-N nylon m e m b r a n e (Amersham, Stockholm). The DNA was cross-linked to the m e m b r a n e by UV irradiation for 2 minutes.
Multilocus probes 33.15 and 33.6. The h y b r i d i z a t i o n protocol was performed according to the m e t h o d of Vassart et al. [20]. The m e m b r a n e with the separated DNA fragments was p r e h y b r i d i z e d for at least 2 hours in the h y b r i d i z a t i o n solution A (40% formamide, 6 x SSC, 5 m M EDTA, 0.25% dried milk). The m e m b r a n e was then h y b r i d i z e d to 32p_ labeled minisatellite m u l t i l o c u s probes 33.15 or 33.6 in the h y b r i d i z a t i o n solution (80/~l/cm 2) at 42°C for at least 24 hours. The minisatellite m u l t i l o c u s probes 33.15 and 33.6 were labeled using a modified r a n d o m p r i m e r extension m e t h o d according to Imperial Chemical Industries PLC with [c~-32p]dGTP (3,000 Ci/mmol). The 32p-labeled probe was purified on a S e p h a d e x G-50 (Pharmacia, Stockholm) column. The 32p-labeled probe was h e a t - d e n a t u r e d before it was a d d e d to the h y b r i d i z a t i o n buffer. After the hybridization, the m e m b r a n e was w a s h e d twice in w a s h i n g solution A1 (2 x SSC, 0.1% SDS) for 15 minutes. T h e n the m e m b r a n e was stringency-washed twice for 15 m i n u t e s at 60°C in washing solution A1. The filter was then rinsed for 15 minutes at room t e m p e r a t u r e in 1 x SSC. The hybridized m e m b r a n e was sealed in a plastic bag and autoradiographed with intensifying screens in a cassette together with x-ray film for 3 - 1 4 days at - 70°C. The film was develo p e d and later analyzed with a laser d e n s i t o m e t e r (LKB, Stockholm).
Single-locus probe XMS1. H y b r i d i z a t i o n w i t h the XMS1 probe was performed according to A m e r s h a m ' s protocol. The m e m b r a n e was p r e h y b r i d i z e d for at least 2 hours at 65°C in h y b r i d i z a t i o n solution B (5 x SSPE, 5 x Denhardt's solution, 0.5% SDS, 0.02 mg/ml d e n a t u r e d herring s p e r m DNA. 5 x SSPE: 0.9 M NaC1, 0.05 M s o d i u m phosphate, 5 m M EDTA pH 7.7). The nylon membrane was then h y b r i d i z e d at 65°C for more than 16 hours with 32p-labeled single-locus probe XMS1. The single-locus minisatellite probe was labeled using the r a n d o m primer extension m e t h o d ( A m e r s h a m ' s m u l t i p r i m e labeling system RPN-1600) with [a-32p] dGTP (3,000 Ci/mmol). After hybridization, the n y l o n membrane was washed twice in washing solution B1 (2 x SSPE, 0.1% SDS) for 10 minutes at room temperature. Then the n y l o n membrane was w a s h e d as follows: 15 minutes at 65°C in solution B2 (1 x SSPE, 0.1% SDS) and twice for 15 minutes at 65°C in solution B3 (0.2 x SSPE, 0.1% SDS); finally, the nylon m e m b r a n e was rinsed in 1 x SSC. The nylon membrane was a u t o r a d i o g r a p h e d with intensifying screens for 1-14 days at - 70°C".
RESULTS The DNA fingerprints from the 22 b l a d d e r t u m o r patients analyzed with the m u l t i l o c u s probes s h o w e d that in 10 of the patients, band alterations were v i s u a l i z e d either as intensity shifts or as changes in n u m b e r of bands (Table 1). In all DNA fingerprint analyses performed, the t u m o r DNA
DNA Alterations in Human Bladder Tumors
Table 1
55
C h a r a c t e r i s t i c s of 22 t u m o r s of t h e b l a d d e r No. of bands with alterations a
Patient
M/F/Age (yr)
7 9 11 13 16 17 19 20 22 24 3 6 12 21 5 10 2 14 8 15 18 23
M/74 M/80 M/70 M/64 M/30 F/61 M/63 M/55 M/69 F/51 M/74 M/64 F/67 M/69 M/88 M/75 F/74 M/79 F/49 M/31 M/59 F/85
Stage
Loss of band
Grade
Ta Ta Ta Ta Ta Ta Ta Ta Ta Ta Ta Ta Ta Ta T1 T1
G1 G1 G1 G2 G1 G~ Cl G1
. .
G1
--
G1 G2 G2 G2
. .
G3
.
T3
G3
.
T3
G3
--
T3
c
b .
T4 e
C2
--f
. .
. .
3 . . .
-1 -. .
1 1 .
.
.
. .
2
----
2 1
.
-1 . 2 1
. --.
. . .
.
-1
1 MO
. . ---
3 2
G3
2 . . . -1 1 . . 1 1
. . .
1 1
Intensity increase in band
. .
--
. . .
G2
Intensity decrease in band
New band
--2 --
. . .
. .
Ploidy 2C 2C 2C 2C 2C 2C 2C 3.8C 2C 2C 2C 2C 2C 3.6C 2C 3.4C 3C 3.2C 3.4C 2.2C 2C 2.4C
Aneuploial cells (%)
88
96 52 22 76 43 2O 14 23
S-phase cells (%} 8 8.2 8.4 11.3 8.3 4.6 5.6 11.8 8.7 4.8 6 13.1 5.6 17.2 6.2 27 NE 37 NE 12 4.7 11
Primary/ recurrent P P P P P P P P P P
R(8410) R(8903) P
R(8808) P P P P P P
Clinical and morphologic data as well as results of flow cytometry and DNA fingerprinting are summarized. a Number of bands scored with all four enzyme/probe combinations. b Squamous cell carcinoma, poorly differentiated. c Adenocarcinoma, well differentiated. d Urethra, not invasive. e Prostate carcinoma. f Malignant lymphoma.
from each patient was compared with the normal constitut i v e D N A (Fig. 1). M o s t of t h e b a n d a l t e r a t i o n s i n t h e b l a d d e r t u m o r s w e r e a s s o c i a t e d w i t h H i n f I e n z y m e d i g e s t i o n of t h e DNA, e s p e c i a l l y w h e n a n a l y z e d w i t h p r o b e 33.15 (Table 2 a n d Fig. 2). A s s h o w n i n T a b l e 3, c h a n g e s a n a l y z e d w i t h p r o b e 33.6 w e r e d e t e c t e d o n l y w i t h H i n f I i n four of t h e seven patients, whereas two were detected only with HaeIII. O n e p a t i e n t ( p a t i e n t 20) s h o w e d c h a n g e s w i t h b o t h t h e r e s t r i c t i o n e n z y m e s . In five p a t i e n t s ( p a t i e n t s 1 0 - 1 2 , 14, a n d 20) b a n d a l t e r a t i o n s w e r e o b s e r v e d w i t h b o t h multilocus probes. New bands were observed only in two p a t i e n t s w h e r e a s loss of b a n d s w a s o b s e r v e d i n eight, i.e. 8 0 % of t h e s a m p l e s f r o m t h e 10 p a t i e n t s . I n t e n s i t y s h i f t s w e r e also o b s e r v e d i n s e v e n of t h e s e 10 p a t i e n t s w i t h a n a l t e r e d t u m o r D N A f i n g e r p r i n t . I n t e n s i t y d e c r e a s e s i n part i c u l a r w e r e o b s e r v e d i n d i f f e r e n t b a n d s i n t h e s e v e n patients. T h e 22 b l a d d e r t u m o r p a t i e n t s w e r e also s t u d i e d w i t h t h e h u m a n s i n g l e - l o c u s p r o b e )~MS1. T h e )~MS1 p r o b e is s p e c i f i c for t h e D I S 7 l o c u s o n c h r o m o s o m e l p [21]. T h e r e s u l t of t h e D N A f i n g e r p r i n t a n a l y s e s w i t h t h i s p r o b e s h o w e d t h a t o n l y o n e p a t i e n t ( p a t i e n t 14) h a d a n a l t e r e d
tumor DNA fingerprint. The DNA fingerprints from this p a t i e n t i m p l y t h a t t h e t u m o r D N A (T) c o n t a i n s t w o d i f f e r e n t p o p u l a t i o n s (Fig. 3), o n e w i t h n o r m a l D N A a n d t h e o t h e r w i t h a l t e r e d DNA. T h e a l t e r a t i o n is a 0 . 9 & i l o b a s e (kb) d e l e t i o n . T h e r e s u l t i n g D N A f i n g e r p r i n t (T) t h u s s h o w s t h e n o r m a l 7.4-kb b a n d a n d a n a d d i t i o n a l 6.5°kb b a n d . T h e c l i n i c a l a n d m o r p h o l o g i c d a t a as w e l l as t h e r e s u l t s of flow c y t o m e t r y are also s h o w n i n T a b l e 1. S i x of t h e 14 n o n i n v a s i v e T a t u m o r s of t h e b l a d d e r h a d D N A b a n d a l t e r a t i o n s , w h e r e a s f o u r of t h e five T 1 - T 3 t u m o r s h a d s u c h a l t e r a t i o n s . N o t a b l y , f o u r of t h e 10 l o w - m a l i g n a n t g r a d e 1 tumors had band alterations, and highly aggressive poorly differentiated aneuploid grade 3 tumors obviously can lack a l t e r a t i o n s . W e n o t e d n e i t h e r a n y c o r r e l a t i o n to p l o i d y or t h e f r a c t i o n of p r o l i f e r a t i n g S - p h a s e c e l l s n o r to r e c u r r e n c e rate of t h e t u m o r s d u r i n g t h e m e a n f o l l o w - u p t i m e of 12 months. The invasive prostate carcinoma and the malignant lymphoma showed no band alterations. DISCUSSION O u r f i n d i n g s c o n f i r m t h e o b s e r v a t i o n s of T h e i n et al. [11], A r m o u r et al. [13], a n d W h i t e et al. [14] t h a t D N A f i n g e r p r i n t
T B
A
. . . .
7~
F i g u r e I DNA fingerprints from bladder tumor tissues: two examples from patient 20 with use of probe 33.15 (A) and 33.6 (B) in HinfI-cut DNA. Patterns with no change in band intensity (A) and decreased band intensity (B) are shown (arrows). Densitometric profiles of the DNA fingerprints are also shown. T, fingerprint from tumor DNA; B, fingerprint from blood DNA.
~ii~ii~!i!~!i! iii~iii:i~i~ijiiii~;~ ~I~
:~%~!i! ~!!i~iiii ii ii~i~i~!~?: ~
: i iii:i~:i~:i~I:
T B
B
CJ1 (3)
D N A A l t e r a t i o n s in H u m a n B l a d d e r T u m o r s
Table 2
57
B a n d a l t e r a t i o n s in D N A fingerprints o b s e r v e d w i t h probe 33.15 w i t h t w o d i f f e r e n t r e s t r i c t i o n e n z y m e s in D N A from seven bladder tumor patients
HinfI Loss of band
5
Intensity decrease
Loss of band
Intensity increase
New band
Altered DNA fragment size (kb)
Patient 11 6 12
New band
HaeIII
2.7 23.5 3.6 -
-
----
-9.2 --
- -
- -
Intensity decrease
Intensity increase
Altered DNA fingerprint size (kb) 4 . 0
.
5.9
--
-
-
.
.
1 9 . 0
.
--
--
- -
-
-
8.6 -
-
13.0 10.0 10 14 8
3.6 -23.5
4.1 ---
-4.1 17.0
Abbreviation: kb, kilobases.
Possible genes affected by p r i m a r y c h r o m o s o m e c h a n g e s h a v e also b e e n d e s c r i b e d [23]. T h e c-abl p r o t o o n c o g e n e , w i t h l o c a l i z a t i o n on c h r o m o s o m e 9q34.1, m a y b e c o m e activ a t e d t h r o u g h a k a r y o t y p i c change. T h e q u e s t i o n of w h e t h e r a specific allelic loss of c h r o m o s o m e 9q is a s s o c i a t e d w i t h the altered D N A fingerprints of the h u m a n b l a d d e r t u m o r s f o u n d in o u r study, m i g h t be a n s w e r e d by use of several c h r o m o s o m e 9 p r o b e s [22]. A n alternate w a y to a n s w e r this q u e s t i o n w i l l be use of a c h r o m o s o m e 9q specific h y p e r v a r i a b l e s i n g l e - l o c u s probe, an a p p r o a c h u s e d earlier by T h e i n et al. [27], w h o o b s e r v e d a c h r o m o s o m a l 7 loss in m y e l o d y s p l a s i a u s i n g an e x t r e m e l y p o l y m o r p h i c D N A probe, p)~g3. H y p o m e t h y l a t i o n of D N A f r o m b o t h b e n i g n a n d malign a n t c o l o n n e o p l a s m s h a v e b e e n r e p o r t e d by G o e l z et al. [28]. T h u s a c h a n g e d D N A m e t h y l a t i o n p a t t e r n of the t u m o r
a n a l y s i s c a n be u s e d to s t u d y s o m a t i c c h a n g e s in t u m o r DNA. T h e result of o u r s t u d y w i t h b l a d d e r t u m o r patients s h o w e d that D N A f i n g e r p r i n t alterations w e r e p r e s e n t in 10 of the 22 e x a m i n e d t u m o r s . T h e m o s t f r e q u e n t alteration d e t e c t e d was a loss of o n e or several bands. T h i s o c c u r r e d in 80% of the altered b l a d d e r tumors. R e c e n t l y , Tsai et al. [22] r e p o r t e d specific allelic losses of c h r o m o s o m e 9, 11, a n d 17 in h u m a n b l a d d e r t u m o r s . T h e greatest f r e q u e n c y of allelic loss was o b s e r v e d in c h r o m o s o m e s 9q (67%) and 17p (63%). Specific allelic loss of c h r o m o s o m e 9q had so far n o t yet b e e n r e l a t e d to any h u m a n cancer. Specific c h r o m o s o m a l c h a n g e s in b l a d d e r c a n c e r w e r e n o t i c e d earlier, h o w e v e r [23-26]. T h e aberrations i n c l u d e d an i s o c h r o m o s o m e of the short arm of c h r o m o s o m e 5, m o n o s o m y of c h r o m o s o m e 9, t r i s o m y of c h r o m o s o m e 7, a n d loss of genes on t h e short arm of c h r o m o s o m e 11 (11p).
Table 3
B a n d alterations in D N A fingerprints o b s e r v e d w i t h probe 33.6 w i t h t w o different r e s t r i c t i o n e n z y m e s in D N A f r o m s e v e n b l a d d e r t u m o r patients
HinfI Loss of band
New band
19 20 22 12 10 14
Intensity decrease
Loss of band
Intensity increase
New band
Altered DNA fragment size (kb)
Patient 11
HaelII
3.1 2.9 . -. 7.4 14.3 --
Abbreviation: kb, kilobases.
-.
. .
. . ---
2.4 2.2
2.3
--
--
--
6.0 .
--
3.4 4.9
-15.0
--3.4
.
. .
4.4 4.1
Intensity increase
Altered DNA fingerprint size (kb)
.
--
Intensity decrease
. .
. . .
. .
. . .
. .
m
m
58
KB
E. Agurell et al.
A
B
BT
BT
23.1 ii i ~ ~
!i!i~i~ii
~ i iii!!!!i: ii :~
9.4 6.5 ....~ :!:i~ fill ii ¸¸ i!~iiiiiii!!iiill
4.3
2.3 2.0 Figure 2 Band alterations observed in two bladder tumor patients. Patient 6 (A) has a loss of one band (23.5 kilobases, kb), one band with decreased intensity (9.2 kb), and one band with increased intensity (5.9 kb). Patient 8 (B) shows loss of one band (23.5 kb) and one with decreased intensity (17.0 kb). The DNA was cut with HinfI and probed with 33.15. Bands with alterations are marked with arrows. T, fingerprint from tumor DNA; B, fingerprint from blood DNA.
could affect DNA fingerprint of a tumor. To clarify the role of methylation, a restriction enzyme that is not sensitive to CpG methylation, i.e., HaeIII or Aluf, can be used. Most of the altered b l a d d e r t u m o r DNA fingerprints in our study, seven of 10 DNA fingerprints, were obtained w h e n HinfI was used to cut the DNA. HinfI is a CpG methylation-sensitive restriction enzyme. W h e n HaeIII was used, five of the 10 altered DNA fingerprints could be detected. Thus, theoretically, a changed methylation pattern of the tumors could in part be an explanation, but for only half of the altered b l a d d e r tumor DNA fingerprints at most. The other five or more altered b l a d d e r t u m o r DNA fingerprints result from genomic alterations other than m e t h y l a t i o n changes. Conventional c h r o m o s o m e analysis has shown abnor-
malities in most b l a d d e r carcinomas irrespective of grade and stage [29, 30], but gross c h r o m o s o m e a b n o r m a l i t i e s w h i c h also can be detected by quantitative cellular DNA analyses have been s h o w n to be associated with m u c h higher aggressiveness of the tumors than n e a r - d i p l o i d ones [17]. The present study c o m p r i s e s only a l i m i t e d n u m b e r of various types of b l a d d e r tumors, but DNA fingerprint alterations are not necessarily related to other t u m o r criteria k n o w n to reflect aggressiveness of the tumors. The question of w h e t h e r DNA alterations as s h o w n by fingerprint analyses are related to clinical m a l i g n a n c y requires experience with a large n u m b e r of various types of tumors and longterm follow-up, however. In this context, White et al. [14] were able to detect fingerprint alterations in five of 12 cases of h y p e r p l a s i a of the prostate and in 12 of 14 in m a l i g n a n t tumors of the prostate. In the o n l y prostate cancer (with invasion into the bladder) s t u d i e d in our series we could not detect abnormalities. The nature of alterations observed in the present material cannot be stated w i t h o u t further m o l e c u l a r analysis. A n e u p l o i d y constitutes a possible error w h e n one interprets DNA fingerprints because it c o u l d have an effect in particular on bands intensity. The p l o i d y analysis s h o w e d that such intensity changes occurred in d i p l o i d cells just as well, however, and furthermore could c o m p r i s e both increase and decrease of intensity in the same t u m o r material. Therefore a n e u p l o i d y is not i n v o l v e d to any large extent. The background of changes of m i n i s a t e l l i t e fingerprints can be elucidated by investigations of i n d u c t i o n of such changes by different c h e m i c a l mutagens in e x p e r i m e n t a l systems. Such studies on mice, Drosophila, and yeast are in progress. Solid tumors consist of a large n u m b e r of subclones, often with heterogenous genomic variation. Occurrence of a particular genetic change in the subclones and in metastases from a p r i m a r y tumor can be used to trace the time of i n d u c t i o n of the change in question: if it i m p l i e s a very early event it can be expected to occur throughout the tumor tissues. On the other hand, if m u t a t e d subclones contribute only to a small a m o u n t of the final DNA fingerprint in the present context, these differences might be lost in the background signal [12]. Identical DNA fingerprints from various tumor localizations in the same patient were described by Smit et al. [31], indicating that the change was an early event in the t u m o r formation. This patient had bilateral ovarian cancer and an uterine m a l i g n a n t mesodermal m i x e d tumor with ascites and metastatic disease. The tumor DNA fingerprints from this patient were altered as c o m p a r e d with the patient's constitutional DNA fingerprint. A possible way to overcome this particular p r o b l e m with tumor heterogeneity is to use a FACS. The FACS technique could sort the different a n e u p l o i d s u b p o p u l a t i o n s that might be observed in the tumor. The different s u b p o p u l a tions could then be analyzed separately by the DNA fingerprint technique to d e t e r m i n e w h e t h e r some relationships exist between altered DNA fingerprints and gross chromosome aberrations.
D N A A l t e r a t i o n s in H u m a n B l a d d e r T u m o r s
59
T B
: ~:
!!i~N~ii¸!!i~,
I
Figure 3
DNA fingerprints from bladder tumor patient 14. The DNA was cut with HinfI and probed with kMS1. Densitometric profiles of the DNA fingerprints are also shown. Band alterations are marked with arrows. T, fingerprint from tumor DNA; B, fingerprint from blood DNA. Lane B has two bands of 7.5 and 4.0 kb and T, in addition to these two bands, also has a new band at 6.5 kb. The intensities of the two bands, (7.4 and 6.5 kb) in T are each approximately 50% of the intensity of the 7.5-kb band in B. The authors thank Professor A. J. Jeffreys (Leicester, England) for providing multilocus probes 33.15 and 33.6 and single-locus probe )~MS1. The work was supported financially by the Swedish Environment Protection Agency and OK Environment Foundation.
REFERENCES 1. Ramel C (1990): Mutation spectrum in carcinogenicity. In: Mechanisms of Environmental Mutagenesis-Carcinogenesis, A Kappas, ed. Plenum Press, New York, pp. 3-24. 2. Ramel C (1988): Short-term testing--are we looking at wrong endpoints? Mutat Res 205:13-24. 3. Jeffreys AJ, Wilson V, Thein SL (1985): Hypervariable 'minisatellite' regions in human DNA. Nature 314:67-73. 4. Jeffreys AJ, Wilson V, Thein SL (1985): Individual-specific fingerprints of human DNA. Nature 316:76-79. 5. Jeffreys AJ, Morton DB (1987): DNA fingerprints of dogs and cats. Animal Genet 18:1-15. 6. Jeffreys AJ, Royle NJ, Wilson V, Wong Z (1988): Spontaneous mutation rates to new length alleles at tandem-repetitive hypervariable loci in human DNA. Nature 332:278-281. 7. Jeffreys AJ, Wilson V, Thein SL, Weatherall DJ, Ponder BAJ (1986): DNA "fingerprints" and segregation analysis of multiple markers in human pedigrees. Am J Hum Genet 39:11-24. 8. Gill P, Jeffreys AJ, Werrett DJ (1985): Forensic application of DNA 'fingerprint'. Nature 318:577-579. 9. Burke T, Bruford MW (1987): DNA fingerprinting in birds. Nature 327:149-152. 10. Wetton JH, Carter RE, Parkin DT, Waiters D (1987): Demographic study of a wild house sparrow population by DNA fingerprinting. Nature 327:147-149.
11. Thein SL, Jeffreys AJ, Gooi HC, Cotter F, Flint J, O'Connor NTJ, Weatherall DJ, Wainscoat JS (1987): Detection of somatic changes in human cancer DNA by DNA fingerprint analysis. Br J Cancer 55:353-356. 12. de Jong D, Voetdijk BMH, Kluin-Nelemans JC, van Ommen GJB, Kluin PhM (1988): Somatic changes in B-lymphoproliferative disorders (B-LPD) detected by DNA fingerprinting. Br J Cancer 58:773-775. 13. Armour JAL, Patel I, Thein SL, Fey MF, Jeffreys AJ (1989): Analysis of somatic mutations at human minisatellite loci in tumors and cell lines. Genomics 4:328-334. 14. White JJ, Neuwirth H, Miller CD, Schneider EL (1990): DNA alterations in prostatic adenocarcinoma and benign prostatic hyperplasia: Detection by DNA fingerprint analyses. Mutat Res 237:37-43. 15. Hermanek P, Sobin LH (1987): TNM Classification of Malignant Tumors, 4th Ed. Springer Verlag, New York. 16. Mostofi FK, Sobin LH, Torloni H (1973): Histological Typing of Urinary Bladder Tumors, International Histological Classification of Tumors 10. World Health Organization, Geneva. 17. Tribukait B (1987): Flow cytometry in assessing the clinical aggressiveness of genito-urinary neoplasms. World J Urol 5:108-122. 18. Sambrook J, Fritsch EF, Maniatis T (1989): Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 19. Southern EM (1975): Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503-527. 20. Vassart G, Georges M, Monsieur R, Brocas M, Lequarre AS, Christophe D (1987): A sequence in M13 phage detects hypervariable minisatellites in human and animal DNA. Science 235:683-684.
60
21. Royle NJ, Clarkson RE, Wong Z, Jeffreys AJ (1988): Clustering of hypervariable minisatellites in the proterminal regions of human autosomes. Genomics 3:352-360. 22. Tsai YC, Nichols PW, Hiti AL, Williams Z, Skinner DG, Jones PA (1990): Allelic losses of chromosomes 9, 11, and 17 in human bladder cancer. Cancer Res 50:44-47. 23. Sandberg AA (1986): Chromosome changes in bladder cancer: Clinical and other correlations. Cancer Genet Cytogenet 19:163-175. 24. Gibas Z, Prout GR Jr, Connolly JG, Pontes JE, Sandberg A (1984): Nonrandom chromosomal changes in transitional cell carcinoma of the bladder. Cancer Res 44:1257-1264. 25. Gibas Z, Prout GR, Pontes JE, Connolly JG, Sandberg AA (1986): A possible specific chromosome change in transitional cell carcinoma of the bladder. Cancer Genet Cytogenet 19:229-238. 26. Fearon ER, Feinberg AP, Hamilton SH, Vogelstein B (1985): Loss of genes on the short arm of chromosome 11 in bladder cancer. Nature 318:377-380.
E. Agurell et al.
27. Thein SL, Oscier DG, Jeffreys AJ, Hesketh C, Pilkington S, Summers C, Fitchett M, Wainscoat JS (1988): Detection of chromosomal 7 loss in myelodysplasia using an extremely polymorphic DNA probe. Br J Cancer 57:131-134. 28. Goelz SE, Vogelstein B, Hamilton SR, Feinberg AP (1985): Hypomethylation of DNA from benign and malignant colon neoplasms, Science 228:187-190. 29. Sandberg AA (1977): Chromosome markers and progression in bladder cnacer. Cancer Res 37:222-229. 30. Tribukait B, Granberg-C)hman I, Wijkstr6m H (1986): Flow cytometric DNA and cytogenetic studies in human tumors: A comparison and discussion of the differences in modal values obtained by the two methods. Cytometry 7:194-199. 31. Smit VTHBM, Cornelisse CJ, de Jong D, Dijkshoorn NJ, Peters AAW, Fleuren GJ (1988): Analysis of tumor heterogeneity in a patient with synchronously occurring female genital tract malignancies by DNA flow cytornetry, DNA fingerprinting, and immunohistochemistry. Cancer 62:1146-1152.
DNA fingerprint analyses were used to examine the constitutional and tumor DNA from 22 bladder tumor patients. DNA alterations, such as loss of bands, new bands, and intensity shifts were observed in 10 of the 22 patients. The most frequent DNA alteration, occurring in 80% of the patients, was a complete loss of one or several bands. Fingerprint abnormalities were present both in lowmalignant superficial tumors and in high-malignant invasive tumors, but were also lacking in the latter group. Apparently no relationship exists between fingerprint abnormalities and gross chromosomal aberrations or the proportion of S-phase cells as measured by flow cytometry or development of recurrent tumors during a limited observation period. Thus, whether fingerprint aberrations express genetic alterations directly involved in the malignancy potential of bladder carcinoma remains an open question.
ABSTRACT:
INTRODUCTION
Development of tumors is a multistep process, which usually implies a series of mutational changes that can include almost all conceivable mutational events [1]. In mutation research of cancer induction and in genetic toxicology in general, emphasis has been placed on conventional endpoints, i.e., point mutations and chromosomal aberration, whereas other changes, such as gene amplification, genetic recombination, and insertion mutations have received far less attention [2]. Highly repetitive DNA sequences are of interest from this viewpoint. Alterations of such a system should include endpoints other than point mutations and chromosomal aberrations, in particular gene amplification and somatic recombination. An analysis of such alterations during tumorigenicity may help shed light on the mutational process during cancer development. Hypervariable minisatellite DNA constitutes a system suitable for investigations of such alterations. These highly repetitive DNA sequences are dispersed throughout the human genome in different polymorphic loci. The polymorphism at these loci are the result of variations of the number of tandem repeats of a short common core se-
From the Department of Genetic and Cellular Toxicology, Wallenberg Laboratory (E. A., C. R.); Department of Genetics (R. L., U. R.), Stockholm University; Department of Urology, South Hospital (U. N.); and Department of Medical Radiobiology, Karolinska Institute (B. T.), Stockholm, Sweden. Address reprint requests to Eva Agurell, Department of Genetic and Cellular Toxicology, Wallenberg Laboratory, Stockholm University, S-106 91 Stockholm, Sweden. Received August 19, 1991; accepted January 27, 1992. © 1992 Elsevier Science Publishing Co., Inc. 655 Avenueof the Americas, New York,NY 10010
quence. These hypervariable minisateUite sequences have been extensively studied and characterized by Jeffreys et al. [3, 4]. The techniques used are Southern analyses of restriction enzyme-treated human DNA after gel electrophoresis in combination with 32p-labeled minisatellite probes. The two multilocus probes 33.15 and 33.6, with a similar core sequence, have been used to produce individual specific DNA fingerprints in humans and animals [5]. Several human single-locus probes were also developed by Jeffreys et al. [6] to study spontaneous mutation rates at individual hypervariable loci and to determine a l l e l e frequencies in different populations. DNA fingerprint analyses have been used to establish sibships in human pedigrees [7], to clarify paternity disputes [4], in forensic work [8], and in analyses of animal populations [9, 10]. Recently, several investigators examined a variety of tumors to study DNA alterations in tumor tissues with the DNA fingerprint technique [11-14]. They noted altered DNA fingerprint patterns in 30-85% of the tumors. We describe the results of 22 tumors of the bladder studied by DNA fingerprint analysis. Cell morphologic and ploidy characterization of the tumors was also performed. The relation between the altered DNA fingerprint patterns and the result of the cell morphologic and ploidy examination are discussed. MATERIALS AND METHODS Patients and Tumors
Tissues from 22 consecutive patients (mean age of 65 years) referred for tumors of the bladder were obtained by transurethal resection. Tumor stage and grade were evaluated ac53 Cancer Genet Cytogenet60:53-60 (1992) 0165-4608/92/$05.0O
54
cording to the criteria of the UICC and the W o r l d Health Organization (WHO) [16]. Eighteen of the tumors were transitional cell carcinomas, one was a squamous cell carcinoma, and one was an adenocarcinoma. One was a T4 a d e n o c a r c i n o m a of the prostate, and one was identified as a malignant l y m p h o m a . Part of the material in saline was p r e p a r e d for DNA flow cytometry as described in detail p r e v i o u s l y [17]. The tumors were s u b d i v i d e d according to their p l o i d y into d i p l o i d (2c) and a n e u p l o i d of various degree. In addition, the p r o p o r t i o n of cells in S-phase was evaluated.
Preparation of DNA Blood. DNA from leukocytes of the h e p a r i n i z e d blood was p r e p a r e d according to the m e t h o d of Jeffreys et al. [4]. Tumor tissue. T u m o r DNA was prepared according to the m e t h o d of Sambrook et al. [18] with m i n o r modifications. Bladder tumors were kept at - 2 0 ° C until the DNA was prepared. The solid tumor tissue was m i n c e d and susp e n d e d in buffer A (10 mM Tris-HC1 pH 8.0, 10 mM EDTA pH 8.0, and 10 mM NaC1; E. Merck, Darmstadt, Germany). The tumor s u s p e n s i o n was w a s h e d once with buffer A and was then r e s u s p e n d e d in buffer A. S o d i u m dodecyl sulphate (SDS, Sigma Chemical, St. Louis, MO) was a d d e d to a final concentration of 0.5%, and proteinase K (Sigma) was a d d e d to a final concentration of 100/~g/ ml. The mixture was incubated for 16 hours at 37°C with shaking; the mixture was then extracted with p h e n o l : chloroform : i s o a m y l a l c o h o l (PCI) 24 : 24 : 1. RNase A (Sigma) was a d d e d to a final concentration of 40 ~g/ml to the water phase, and the mixture was incubated at 37°C with shaking; after 1 hour, SDS (final concentration 0.5%) and proteinase K (final concentration 100 /~g/ml) were a d d e d to the tube. After 1-hour incubation at 37°C with shaking, the mixture was extracted twice with PCI and once with chloroform. The DNA content in the samples was d e t e r m i n e d with an ultraviolet (UV) s p e c t r o p h o t o m e t e r by reading the absorbance at 260 nm. The purity of the samples was checked by the ratio of the absorbance at 260 n m and 280 nm.
DNA Analysis DNA from both normal and t u m o r tissue of each patient was digested with restriction enzymes HinfI or HaeIII (Betheseda Research Laboratories, BRL, Gaithersburg, MD) according to the m e t h o d of Jeffreys et al. [4]. Ten micrograms DNA was digested for 16 hours at 37°C with HinfI or HaeIlI with a d d i t i o n of 4 m M s p e r m i d i n e (United States Biochemical, Cleveland, OH). The s p e r m i d i n e was a d d e d to aid in the cutting of DNA. DNA was extracted with PCI and chloroform and recovered by ethanol precipitation. The resulting DNA fragments of each patient, normal and t u m o r DNA, were run side by side in a 20-cm-long 0.8% agarose (Bethesda Research Laboratories) gel in 1 x TBE (89 mM trisborate, 89 m M boric acid, 2 mM EDTA) for 24 hours at 50 V at room temperature. The separated DNA fragments were transferred according to the method of Southern [19] in 10 x SSC) 1.5 M NaC1, 0.15 M trisodium
E. Agurell et al. citrate) to a Hybond-N nylon m e m b r a n e (Amersham, Stockholm). The DNA was cross-linked to the m e m b r a n e by UV irradiation for 2 minutes.
Multilocus probes 33.15 and 33.6. The h y b r i d i z a t i o n protocol was performed according to the m e t h o d of Vassart et al. [20]. The m e m b r a n e with the separated DNA fragments was p r e h y b r i d i z e d for at least 2 hours in the h y b r i d i z a t i o n solution A (40% formamide, 6 x SSC, 5 m M EDTA, 0.25% dried milk). The m e m b r a n e was then h y b r i d i z e d to 32p_ labeled minisatellite m u l t i l o c u s probes 33.15 or 33.6 in the h y b r i d i z a t i o n solution (80/~l/cm 2) at 42°C for at least 24 hours. The minisatellite m u l t i l o c u s probes 33.15 and 33.6 were labeled using a modified r a n d o m p r i m e r extension m e t h o d according to Imperial Chemical Industries PLC with [c~-32p]dGTP (3,000 Ci/mmol). The 32p-labeled probe was purified on a S e p h a d e x G-50 (Pharmacia, Stockholm) column. The 32p-labeled probe was h e a t - d e n a t u r e d before it was a d d e d to the h y b r i d i z a t i o n buffer. After the hybridization, the m e m b r a n e was w a s h e d twice in w a s h i n g solution A1 (2 x SSC, 0.1% SDS) for 15 minutes. T h e n the m e m b r a n e was stringency-washed twice for 15 m i n u t e s at 60°C in washing solution A1. The filter was then rinsed for 15 minutes at room t e m p e r a t u r e in 1 x SSC. The hybridized m e m b r a n e was sealed in a plastic bag and autoradiographed with intensifying screens in a cassette together with x-ray film for 3 - 1 4 days at - 70°C. The film was develo p e d and later analyzed with a laser d e n s i t o m e t e r (LKB, Stockholm).
Single-locus probe XMS1. H y b r i d i z a t i o n w i t h the XMS1 probe was performed according to A m e r s h a m ' s protocol. The m e m b r a n e was p r e h y b r i d i z e d for at least 2 hours at 65°C in h y b r i d i z a t i o n solution B (5 x SSPE, 5 x Denhardt's solution, 0.5% SDS, 0.02 mg/ml d e n a t u r e d herring s p e r m DNA. 5 x SSPE: 0.9 M NaC1, 0.05 M s o d i u m phosphate, 5 m M EDTA pH 7.7). The nylon membrane was then h y b r i d i z e d at 65°C for more than 16 hours with 32p-labeled single-locus probe XMS1. The single-locus minisatellite probe was labeled using the r a n d o m primer extension m e t h o d ( A m e r s h a m ' s m u l t i p r i m e labeling system RPN-1600) with [a-32p] dGTP (3,000 Ci/mmol). After hybridization, the n y l o n membrane was washed twice in washing solution B1 (2 x SSPE, 0.1% SDS) for 10 minutes at room temperature. Then the n y l o n membrane was w a s h e d as follows: 15 minutes at 65°C in solution B2 (1 x SSPE, 0.1% SDS) and twice for 15 minutes at 65°C in solution B3 (0.2 x SSPE, 0.1% SDS); finally, the nylon m e m b r a n e was rinsed in 1 x SSC. The nylon membrane was a u t o r a d i o g r a p h e d with intensifying screens for 1-14 days at - 70°C".
RESULTS The DNA fingerprints from the 22 b l a d d e r t u m o r patients analyzed with the m u l t i l o c u s probes s h o w e d that in 10 of the patients, band alterations were v i s u a l i z e d either as intensity shifts or as changes in n u m b e r of bands (Table 1). In all DNA fingerprint analyses performed, the t u m o r DNA
DNA Alterations in Human Bladder Tumors
Table 1
55
C h a r a c t e r i s t i c s of 22 t u m o r s of t h e b l a d d e r No. of bands with alterations a
Patient
M/F/Age (yr)
7 9 11 13 16 17 19 20 22 24 3 6 12 21 5 10 2 14 8 15 18 23
M/74 M/80 M/70 M/64 M/30 F/61 M/63 M/55 M/69 F/51 M/74 M/64 F/67 M/69 M/88 M/75 F/74 M/79 F/49 M/31 M/59 F/85
Stage
Loss of band
Grade
Ta Ta Ta Ta Ta Ta Ta Ta Ta Ta Ta Ta Ta Ta T1 T1
G1 G1 G1 G2 G1 G~ Cl G1
. .
G1
--
G1 G2 G2 G2
. .
G3
.
T3
G3
.
T3
G3
--
T3
c
b .
T4 e
C2
--f
. .
. .
3 . . .
-1 -. .
1 1 .
.
.
. .
2
----
2 1
.
-1 . 2 1
. --.
. . .
.
-1
1 MO
. . ---
3 2
G3
2 . . . -1 1 . . 1 1
. . .
1 1
Intensity increase in band
. .
--
. . .
G2
Intensity decrease in band
New band
--2 --
. . .
. .
Ploidy 2C 2C 2C 2C 2C 2C 2C 3.8C 2C 2C 2C 2C 2C 3.6C 2C 3.4C 3C 3.2C 3.4C 2.2C 2C 2.4C
Aneuploial cells (%)
88
96 52 22 76 43 2O 14 23
S-phase cells (%} 8 8.2 8.4 11.3 8.3 4.6 5.6 11.8 8.7 4.8 6 13.1 5.6 17.2 6.2 27 NE 37 NE 12 4.7 11
Primary/ recurrent P P P P P P P P P P
R(8410) R(8903) P
R(8808) P P P P P P
Clinical and morphologic data as well as results of flow cytometry and DNA fingerprinting are summarized. a Number of bands scored with all four enzyme/probe combinations. b Squamous cell carcinoma, poorly differentiated. c Adenocarcinoma, well differentiated. d Urethra, not invasive. e Prostate carcinoma. f Malignant lymphoma.
from each patient was compared with the normal constitut i v e D N A (Fig. 1). M o s t of t h e b a n d a l t e r a t i o n s i n t h e b l a d d e r t u m o r s w e r e a s s o c i a t e d w i t h H i n f I e n z y m e d i g e s t i o n of t h e DNA, e s p e c i a l l y w h e n a n a l y z e d w i t h p r o b e 33.15 (Table 2 a n d Fig. 2). A s s h o w n i n T a b l e 3, c h a n g e s a n a l y z e d w i t h p r o b e 33.6 w e r e d e t e c t e d o n l y w i t h H i n f I i n four of t h e seven patients, whereas two were detected only with HaeIII. O n e p a t i e n t ( p a t i e n t 20) s h o w e d c h a n g e s w i t h b o t h t h e r e s t r i c t i o n e n z y m e s . In five p a t i e n t s ( p a t i e n t s 1 0 - 1 2 , 14, a n d 20) b a n d a l t e r a t i o n s w e r e o b s e r v e d w i t h b o t h multilocus probes. New bands were observed only in two p a t i e n t s w h e r e a s loss of b a n d s w a s o b s e r v e d i n eight, i.e. 8 0 % of t h e s a m p l e s f r o m t h e 10 p a t i e n t s . I n t e n s i t y s h i f t s w e r e also o b s e r v e d i n s e v e n of t h e s e 10 p a t i e n t s w i t h a n a l t e r e d t u m o r D N A f i n g e r p r i n t . I n t e n s i t y d e c r e a s e s i n part i c u l a r w e r e o b s e r v e d i n d i f f e r e n t b a n d s i n t h e s e v e n patients. T h e 22 b l a d d e r t u m o r p a t i e n t s w e r e also s t u d i e d w i t h t h e h u m a n s i n g l e - l o c u s p r o b e )~MS1. T h e )~MS1 p r o b e is s p e c i f i c for t h e D I S 7 l o c u s o n c h r o m o s o m e l p [21]. T h e r e s u l t of t h e D N A f i n g e r p r i n t a n a l y s e s w i t h t h i s p r o b e s h o w e d t h a t o n l y o n e p a t i e n t ( p a t i e n t 14) h a d a n a l t e r e d
tumor DNA fingerprint. The DNA fingerprints from this p a t i e n t i m p l y t h a t t h e t u m o r D N A (T) c o n t a i n s t w o d i f f e r e n t p o p u l a t i o n s (Fig. 3), o n e w i t h n o r m a l D N A a n d t h e o t h e r w i t h a l t e r e d DNA. T h e a l t e r a t i o n is a 0 . 9 & i l o b a s e (kb) d e l e t i o n . T h e r e s u l t i n g D N A f i n g e r p r i n t (T) t h u s s h o w s t h e n o r m a l 7.4-kb b a n d a n d a n a d d i t i o n a l 6.5°kb b a n d . T h e c l i n i c a l a n d m o r p h o l o g i c d a t a as w e l l as t h e r e s u l t s of flow c y t o m e t r y are also s h o w n i n T a b l e 1. S i x of t h e 14 n o n i n v a s i v e T a t u m o r s of t h e b l a d d e r h a d D N A b a n d a l t e r a t i o n s , w h e r e a s f o u r of t h e five T 1 - T 3 t u m o r s h a d s u c h a l t e r a t i o n s . N o t a b l y , f o u r of t h e 10 l o w - m a l i g n a n t g r a d e 1 tumors had band alterations, and highly aggressive poorly differentiated aneuploid grade 3 tumors obviously can lack a l t e r a t i o n s . W e n o t e d n e i t h e r a n y c o r r e l a t i o n to p l o i d y or t h e f r a c t i o n of p r o l i f e r a t i n g S - p h a s e c e l l s n o r to r e c u r r e n c e rate of t h e t u m o r s d u r i n g t h e m e a n f o l l o w - u p t i m e of 12 months. The invasive prostate carcinoma and the malignant lymphoma showed no band alterations. DISCUSSION O u r f i n d i n g s c o n f i r m t h e o b s e r v a t i o n s of T h e i n et al. [11], A r m o u r et al. [13], a n d W h i t e et al. [14] t h a t D N A f i n g e r p r i n t
T B
A
. . . .
7~
F i g u r e I DNA fingerprints from bladder tumor tissues: two examples from patient 20 with use of probe 33.15 (A) and 33.6 (B) in HinfI-cut DNA. Patterns with no change in band intensity (A) and decreased band intensity (B) are shown (arrows). Densitometric profiles of the DNA fingerprints are also shown. T, fingerprint from tumor DNA; B, fingerprint from blood DNA.
~ii~ii~!i!~!i! iii~iii:i~i~ijiiii~;~ ~I~
:~%~!i! ~!!i~iiii ii ii~i~i~!~?: ~
: i iii:i~:i~:i~I:
T B
B
CJ1 (3)
D N A A l t e r a t i o n s in H u m a n B l a d d e r T u m o r s
Table 2
57
B a n d a l t e r a t i o n s in D N A fingerprints o b s e r v e d w i t h probe 33.15 w i t h t w o d i f f e r e n t r e s t r i c t i o n e n z y m e s in D N A from seven bladder tumor patients
HinfI Loss of band
5
Intensity decrease
Loss of band
Intensity increase
New band
Altered DNA fragment size (kb)
Patient 11 6 12
New band
HaeIII
2.7 23.5 3.6 -
-
----
-9.2 --
- -
- -
Intensity decrease
Intensity increase
Altered DNA fingerprint size (kb) 4 . 0
.
5.9
--
-
-
.
.
1 9 . 0
.
--
--
- -
-
-
8.6 -
-
13.0 10.0 10 14 8
3.6 -23.5
4.1 ---
-4.1 17.0
Abbreviation: kb, kilobases.
Possible genes affected by p r i m a r y c h r o m o s o m e c h a n g e s h a v e also b e e n d e s c r i b e d [23]. T h e c-abl p r o t o o n c o g e n e , w i t h l o c a l i z a t i o n on c h r o m o s o m e 9q34.1, m a y b e c o m e activ a t e d t h r o u g h a k a r y o t y p i c change. T h e q u e s t i o n of w h e t h e r a specific allelic loss of c h r o m o s o m e 9q is a s s o c i a t e d w i t h the altered D N A fingerprints of the h u m a n b l a d d e r t u m o r s f o u n d in o u r study, m i g h t be a n s w e r e d by use of several c h r o m o s o m e 9 p r o b e s [22]. A n alternate w a y to a n s w e r this q u e s t i o n w i l l be use of a c h r o m o s o m e 9q specific h y p e r v a r i a b l e s i n g l e - l o c u s probe, an a p p r o a c h u s e d earlier by T h e i n et al. [27], w h o o b s e r v e d a c h r o m o s o m a l 7 loss in m y e l o d y s p l a s i a u s i n g an e x t r e m e l y p o l y m o r p h i c D N A probe, p)~g3. H y p o m e t h y l a t i o n of D N A f r o m b o t h b e n i g n a n d malign a n t c o l o n n e o p l a s m s h a v e b e e n r e p o r t e d by G o e l z et al. [28]. T h u s a c h a n g e d D N A m e t h y l a t i o n p a t t e r n of the t u m o r
a n a l y s i s c a n be u s e d to s t u d y s o m a t i c c h a n g e s in t u m o r DNA. T h e result of o u r s t u d y w i t h b l a d d e r t u m o r patients s h o w e d that D N A f i n g e r p r i n t alterations w e r e p r e s e n t in 10 of the 22 e x a m i n e d t u m o r s . T h e m o s t f r e q u e n t alteration d e t e c t e d was a loss of o n e or several bands. T h i s o c c u r r e d in 80% of the altered b l a d d e r tumors. R e c e n t l y , Tsai et al. [22] r e p o r t e d specific allelic losses of c h r o m o s o m e 9, 11, a n d 17 in h u m a n b l a d d e r t u m o r s . T h e greatest f r e q u e n c y of allelic loss was o b s e r v e d in c h r o m o s o m e s 9q (67%) and 17p (63%). Specific allelic loss of c h r o m o s o m e 9q had so far n o t yet b e e n r e l a t e d to any h u m a n cancer. Specific c h r o m o s o m a l c h a n g e s in b l a d d e r c a n c e r w e r e n o t i c e d earlier, h o w e v e r [23-26]. T h e aberrations i n c l u d e d an i s o c h r o m o s o m e of the short arm of c h r o m o s o m e 5, m o n o s o m y of c h r o m o s o m e 9, t r i s o m y of c h r o m o s o m e 7, a n d loss of genes on t h e short arm of c h r o m o s o m e 11 (11p).
Table 3
B a n d alterations in D N A fingerprints o b s e r v e d w i t h probe 33.6 w i t h t w o different r e s t r i c t i o n e n z y m e s in D N A f r o m s e v e n b l a d d e r t u m o r patients
HinfI Loss of band
New band
19 20 22 12 10 14
Intensity decrease
Loss of band
Intensity increase
New band
Altered DNA fragment size (kb)
Patient 11
HaelII
3.1 2.9 . -. 7.4 14.3 --
Abbreviation: kb, kilobases.
-.
. .
. . ---
2.4 2.2
2.3
--
--
--
6.0 .
--
3.4 4.9
-15.0
--3.4
.
. .
4.4 4.1
Intensity increase
Altered DNA fingerprint size (kb)
.
--
Intensity decrease
. .
. . .
. .
. . .
. .
m
m
58
KB
E. Agurell et al.
A
B
BT
BT
23.1 ii i ~ ~
!i!i~i~ii
~ i iii!!!!i: ii :~
9.4 6.5 ....~ :!:i~ fill ii ¸¸ i!~iiiiiii!!iiill
4.3
2.3 2.0 Figure 2 Band alterations observed in two bladder tumor patients. Patient 6 (A) has a loss of one band (23.5 kilobases, kb), one band with decreased intensity (9.2 kb), and one band with increased intensity (5.9 kb). Patient 8 (B) shows loss of one band (23.5 kb) and one with decreased intensity (17.0 kb). The DNA was cut with HinfI and probed with 33.15. Bands with alterations are marked with arrows. T, fingerprint from tumor DNA; B, fingerprint from blood DNA.
could affect DNA fingerprint of a tumor. To clarify the role of methylation, a restriction enzyme that is not sensitive to CpG methylation, i.e., HaeIII or Aluf, can be used. Most of the altered b l a d d e r t u m o r DNA fingerprints in our study, seven of 10 DNA fingerprints, were obtained w h e n HinfI was used to cut the DNA. HinfI is a CpG methylation-sensitive restriction enzyme. W h e n HaeIII was used, five of the 10 altered DNA fingerprints could be detected. Thus, theoretically, a changed methylation pattern of the tumors could in part be an explanation, but for only half of the altered b l a d d e r tumor DNA fingerprints at most. The other five or more altered b l a d d e r t u m o r DNA fingerprints result from genomic alterations other than m e t h y l a t i o n changes. Conventional c h r o m o s o m e analysis has shown abnor-
malities in most b l a d d e r carcinomas irrespective of grade and stage [29, 30], but gross c h r o m o s o m e a b n o r m a l i t i e s w h i c h also can be detected by quantitative cellular DNA analyses have been s h o w n to be associated with m u c h higher aggressiveness of the tumors than n e a r - d i p l o i d ones [17]. The present study c o m p r i s e s only a l i m i t e d n u m b e r of various types of b l a d d e r tumors, but DNA fingerprint alterations are not necessarily related to other t u m o r criteria k n o w n to reflect aggressiveness of the tumors. The question of w h e t h e r DNA alterations as s h o w n by fingerprint analyses are related to clinical m a l i g n a n c y requires experience with a large n u m b e r of various types of tumors and longterm follow-up, however. In this context, White et al. [14] were able to detect fingerprint alterations in five of 12 cases of h y p e r p l a s i a of the prostate and in 12 of 14 in m a l i g n a n t tumors of the prostate. In the o n l y prostate cancer (with invasion into the bladder) s t u d i e d in our series we could not detect abnormalities. The nature of alterations observed in the present material cannot be stated w i t h o u t further m o l e c u l a r analysis. A n e u p l o i d y constitutes a possible error w h e n one interprets DNA fingerprints because it c o u l d have an effect in particular on bands intensity. The p l o i d y analysis s h o w e d that such intensity changes occurred in d i p l o i d cells just as well, however, and furthermore could c o m p r i s e both increase and decrease of intensity in the same t u m o r material. Therefore a n e u p l o i d y is not i n v o l v e d to any large extent. The background of changes of m i n i s a t e l l i t e fingerprints can be elucidated by investigations of i n d u c t i o n of such changes by different c h e m i c a l mutagens in e x p e r i m e n t a l systems. Such studies on mice, Drosophila, and yeast are in progress. Solid tumors consist of a large n u m b e r of subclones, often with heterogenous genomic variation. Occurrence of a particular genetic change in the subclones and in metastases from a p r i m a r y tumor can be used to trace the time of i n d u c t i o n of the change in question: if it i m p l i e s a very early event it can be expected to occur throughout the tumor tissues. On the other hand, if m u t a t e d subclones contribute only to a small a m o u n t of the final DNA fingerprint in the present context, these differences might be lost in the background signal [12]. Identical DNA fingerprints from various tumor localizations in the same patient were described by Smit et al. [31], indicating that the change was an early event in the t u m o r formation. This patient had bilateral ovarian cancer and an uterine m a l i g n a n t mesodermal m i x e d tumor with ascites and metastatic disease. The tumor DNA fingerprints from this patient were altered as c o m p a r e d with the patient's constitutional DNA fingerprint. A possible way to overcome this particular p r o b l e m with tumor heterogeneity is to use a FACS. The FACS technique could sort the different a n e u p l o i d s u b p o p u l a t i o n s that might be observed in the tumor. The different s u b p o p u l a tions could then be analyzed separately by the DNA fingerprint technique to d e t e r m i n e w h e t h e r some relationships exist between altered DNA fingerprints and gross chromosome aberrations.
D N A A l t e r a t i o n s in H u m a n B l a d d e r T u m o r s
59
T B
: ~:
!!i~N~ii¸!!i~,
I
Figure 3
DNA fingerprints from bladder tumor patient 14. The DNA was cut with HinfI and probed with kMS1. Densitometric profiles of the DNA fingerprints are also shown. Band alterations are marked with arrows. T, fingerprint from tumor DNA; B, fingerprint from blood DNA. Lane B has two bands of 7.5 and 4.0 kb and T, in addition to these two bands, also has a new band at 6.5 kb. The intensities of the two bands, (7.4 and 6.5 kb) in T are each approximately 50% of the intensity of the 7.5-kb band in B. The authors thank Professor A. J. Jeffreys (Leicester, England) for providing multilocus probes 33.15 and 33.6 and single-locus probe )~MS1. The work was supported financially by the Swedish Environment Protection Agency and OK Environment Foundation.
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