Cytogenetic and Molecular Studies of a Familial Renal Cell Carcinoma Hans-Joachim H. Decker, Bernd Wullich, Jean M. Whaley, Guillermo Herrera, Sabine M. K|auck, Avery A. Sandberg, David W. Yandell, and Bernd R. Seizinger
ABSTRACT: In a previously studied f a m i l y with inherited renal cell carcinoma (RCC), RCC was shown to segregate with a constitutional balanced t(3;8)(p14.2;q24.1). In addition, we recently showed that in a RCC tumor from this f a m i l y the constitutional translocation became unbalanced, suggesting a genetic m e c h a n i s m that m a y be associated with the primary genetic events of tumorigenesis. We now report that the RCC tumor cells from this case showed additional cytogenetic alterations, possibly related to tumor progression, which include an additional tumor-specific translocation involving band 14 of chromosome 13. Because this band contains the retinoblastoma (RB) gene, we e x a m i n e d the tumor for aberrations in the RB gene using DNA sequence p o l y m o r p h i s m analysis and pulsed-field gel electrophoresis (PFGE), but did not detect alterations in the RB gene.
INTRODUCTION In 1979, Cohen et al. described renal cell carcinomas (RCC) in 10 adults from three consecutive generations of an Italian-American family [1]. Constitutional karyotypes of 10 of 22 tested family members showed a balanced translocation between chromosomes 3 and 8 which segregated with the RCCs. Wang and Perkins [2] specified the breakpoints of this reciprocal translocation as t(3;8)(p14.2;q24.1). An RCC of this family was studied with a variety of cytogenetic and molecular genetic techniques. Cytogenetic and restriction fragment length polymorphisms (RFLP) studies [3] demonstrated that the only observed tumor cell clone retained the normal chromosome 3, whereas der(8), carrying the distal part of 3p, was lost. Clinical follow-up and some of the genetic data of this case were reported elsewhere [3]. We report a more detailed analysis of genetic alterations in this RCC, which focused on the RB gene. Loss of genetic material from the short arm of chromosome 3 is the most frequently observed nonrandom aberration in both sporadic and familial RCC, suggesting the exis-
From the Molecular Neuro-Oncology Laboratory, Massachu~ setts General Hospital and Harvard Medical School Boston (H.-J. H. D., J. M. W., S. M. K., B. R. S.), Cancer Center of the Southwest Biomedical Research Institute, Scottsdale, Arizona (B. W., A. A. S.), and Department of Ophthalmalogy, Massachusetts Eye and Ear Infirmary, and Harvard Medical School (G. H., D. W. Y.), Boston, Massachusetts. Address reprint requests to Dr. Hans-Joachim H. Decker, Department of Oncology/Hematology, Medical School of the Johannes-Gutenberg-University Mainz, Langenbeck-Str. 1, D-6500 Mainz, Germany. Received December 12, 1991; accepted April 1, 1992.
tence of one or even more tumor suppressor gene(s) in the 3p region. RFLP analyses in sporadic RCC have shown loss of heterozygosity (LOH) at several chromosomal loci other than 3p, but such changes are less frequent than is LOH of 3p. Of particular note is that 15-33% of sporadic RCC have had LOH of genetic loci from 13q [4-6]. LOH of 13q has been found only in advanced cases of RCC, however; therefore, the RB gene, located at 13q14, was suggested to be involved in RCC tumor progression [5l. Loss of RB function has been described in a large variety of tumor types, such as retinoblastomas [7-9], osteosarcomas [9-11], lung cancers [12-14], sarcomas [10, 15, 16], prostate carcinomas [17, 18], and breast cancers [19], but no direct evidence for consistent RB gene involvement in RCC has yet been reported [20]. Because 13q14 may have been involved in an additional unbalanced translocation in the familial RCC in this study, we performed pulsed-field gel electrophoresis (PFGE) and DNA sequence polymorphism analysis of the RB gene to seek aberrations in this tumor suppressor locus. CASE REPORT AND PATHOLOGY
Bilateral partial nephrectomies were performed to remove asymptomatic renal tumors in a 41-year-old white woman. Twelve years later, the patient developed a renal mass in the remnant of the left kidney. She had received no chemotherapy. Total nephrectomy showed a clear cell RCC, various regions of which were provided for genetic studies. MATERIALS AND METHODS Cell Culture and Cytogenetics A piece of tumor was disaggregated [21] with collagenase (final concentration 400 U/ml) and agitated for 4 hours. The
25 © 1992 Elsevier Science Publishing Co., Inc. 655 A v e n u e of the Americas, New York, NY 10010
Cancer Genet Cytogenet 6 3 : 2 5 - 3 1 (1992J 0165-4608/92/$05.00
26 single cells were seeded immediately into flasks, flaskettes, and directly on coverslips. The tumor cells grew attached in RPMI medium, supplemented with 20% fetal bovine serum, L-glutamine, and antibiotics. Nine harvests were performed: the first after 3 days, and the last after 4 weeks. The best results were obtained by a modified synchronization method [22] and the coverslip in situ harvest procedure [23]. GTG- and CBG-banding were performed according to standard procedures [24, 25].
Fluorescence In Situ Hybridization (FISH), DNA Probes, and Cytochemical Detection FISH was done according to the protocol of Pinkel et al. [26] with modifications [27]. Human c~-satellite DNA probes [28] detecting sequences of the centromeric regions of chromosomes 3 and 16 were used: pa3-5 [D3Z1], and pSE 16[D16Z2]. All probes were purchased in the biotin-labeled form from Oncor (Gaithersburg, MD). The intensity of biotin-linked fluorescence was amplified by adding a layer of biotinylated goat anti-avidin antibody to the first layer before adding another layer of fluorescence-avidin DCS (both purchased from Vector Laboratories, Burlingame, CA). The counterstain was propidium iodide in an antifade solution [29].
DNA Sequence Polymorphism (DSP) in RB 1.20 DNA was extracted from 1) cultured RCC cells, 2) normal kidney tissue, and 3) the primary RCC and subsequently PCR-amplified. The primer pair for amplification was derived for the polymorphic sequence RB 1.20 from the 3' end of exon 20: 5'-TGAATGAACAATTGACTAAA-3'(position 431-450) and 5'-CTCTATCTCAGAGTGAGACAATG3'(position 548-569). One of the primers was 32p endlabeled and used in a polymerization reaction with Sequenase (U.S. Biochemical) and all four deoxynucleotides. The reaction products were heat-denatured, electrophoresed on a 6% polyacrylamide gel, and autoradiographed for 12 hours [30].
PFGE and DNA Probes To yield high-molecular-weight DNA, single suspended cells of each sample (tumor cells from different passages, and leukocytes from normal individuals) were imbedded in agarose blocks as previously described [31]. For restriction digestion, Notl was used, because in the long-range physical map around RB the NotI fragment spans 930 (kilobases), including all 200 kb of the retinoblastoma (RB) gene [32]. PFGE was performed in a clamped homogeneous electric field using the LKB pulsaphor system in basic mode. Charged Hybond N + filters were used. The probes for the RB were gifts from Dr. Thaddeus P. Dryja. For hybridization, probes from the 3' end (p123M1.8) and the 5' end (p68RS2.0) of the RB gene were used.
RESULTS Different fractions of the tumor cells in culture exhibited heterogeneous morphologies. Figure 1 shows fractions (A and F) of the tumor. In contrast to the varying morphology,
H.-J. H. Decker et al. cytogenetic, and molecular analysis showed no difference between these separately harvested fractions. Nine harvests were performed after 3-28 days in culture. There was only a single tumor cell clone: 167 cells showed 43,XX,der(3)t(3;8)(p14.2;q24),- 8 , - 1 3 , - 14, der(16)t(13 ;?;16)(16pter--~ 16q24::?::13q14?13qter). Fifteen cells were hypodiploid with random loss. Twelve cells were hypotetraploid; the number of hypotetraploid cells increased with prolonged culture. After 2 weeks of culture, only two cells were detected with the constitutional karyotype 46,XX,t(3;8)(p14.2;q24). These two cells retained the derivative 8 which was lost in the tumor cell clone. In addition, both homologues of chromosomes 13, 14, and 16 were intact in these two cells. Figure 2 shows a partial karyotype with the derivative chromosomes, der(3) and der(16). CBG-banding proved that the derivative chromosome 16 was not dicentric (data not shown). FISH verified the cytogenetic results, showing that the derivative was stemming from chromosome 16 (Fig. 3d) and that two centromeres of chromosome 3 were retained in 85.5-87% of the cells scored. Two signals from the centromere probes of chromosome 16 were observed in 77-90% of cells monitored. Two hundred seventeen (pa35) and 431 (pSE16) nuclei were screened (Fig. 4). DNA sequence polymorphism analysis of RB after polymerase chain reaction (PCR) amplification (Fig. 5) did not show any differences when cultured RCC cells (lane 1), normal kidney tissue (lane 2), and primary RCC (lane 3) of the patient were compared. Two probes from different regions of the RB gene did not detect structural rearrangements after PFGE (Fig. 6) of NotI-digested DNA of controls and tumor cells from morphologically different cultures (compare to Fig. 1A and F) after 4 and 6 weeks (S) in culture.
DISCUSSION The constitutional t(3;8)(p14.2;q24.1) has been clearly associated with development of RCC in 10 members of one family [1, 3]. We describe the genetic alterations observed in the first RCC of this family accessible for cytogenetic studies: the constitutional translocation became unbalanced due to the loss of the derivative (8) carrying the already altered short arm of chromosome 3. Surprisingly, no aberrations could be detected in the normal homologue 3 of our case by cytogenetic and RFLP studies [3]. The retention of both chromosome 3 centromeres, one of the normal homologue and the other of the der(3), was also verified by FISH techniques. These findings may indicate a more complex mechanism of tumorigenesis than simple loss of both copies of a single tumor-suppressor gene. More than one gene on 3p may be involved in the tumorigenesis of RCC; the yon Hippel Lindau gene is one candidate [33-36]. At the time the experiments were performed, no repetitive probes for chromosome 8 centromere were available. Whereas alterations of DNA sequences from 3p appear to be crucial for tumor initiation in sporadic and familial RCC, little is known about genetic alterations leading to tumor progression in RCCs. For sporadic RCC, evidence
27
Figure I Cells from different regions (A, F) of the RCC grown in culture for 2 weeks: Although they show a clearly different morphology, no difference was evident when they were analyzed separately by cytogenetics of PFGE (as compared with Fig. 2).
Figure 2 Selected GTG-banded partial karyotypes showing the structural unbalanced rearrangements observed in three different metaphases: der(3)t(3;8)(p14.2;q24) and der(16)t(13;?;16)(16pter--~16q24::?::13q14?-~13qter).
\ 16q24
-|/
b _i_~ =k
,/
/
13q14?
8q24
c-i- -l3
d e r (3)
U 8
13
der (16)
16
28
d Figure 3 Representative FISH with biotinylated a-satellite probes for the centromeric regions of chromosomes 3[pa3-5] (a and b) and 16[pSE16] (c and d) in metaphases and interphases. Arrow (c) marks the der(16).
F a m i l i a l RCC
29 100%
1{)0% #16
#3
pa 3-5 ID3ZII
pSE 16 IDI6 Z 2l
50~t
5O%
r 0
I
2
__n 3
>3
?
o
I--I--qr 0
I
2
3
>3
?
Figure 4 Histogram showing the quantitative analysis of FISH: No significant number of cells with loss of the der(3) was noted in two independent investigations (open and hatched columns).
123 Figure 5 DNA sequence polymorphism analysis after PCR amplification: No differences were noted when cultured 1) RCC cells 2) normal kidney tissue, and 3) and the primary RCC of the patient were compared.
shows i n v o l v e m e n t of other c h r o m o s o m a l loci than 3p, but all of these are less frequent than 3p loss [37, 38]. In our case, in a d d i t i o n to c h r o m o s o m e 3 and 8 alterations, one c h r o m o s o m e 14 was lost in all tumor cells. Although m o n o s o m y 14 is c o m m o n in sporadic and familial RCC (30% in - 3 0 0 RCCs and RCC cell lines reported so far), the significance of this is not known, but it could be related to t u m o r progression [39]. In three previous studies [4-6], LOH of DNA fragments from 13q was observed in a d v a n c e d sporadic RCC. This led to the proposal that the RB gene might be altered during tumor progression in RCC [5], but for sporadic RCC, the RB gene was recently shown to be infrequently involved by the means of Southern, Northern, and Western blotting and i m m u n o s t a i n i n g [20]. No m o l e c u l a r studies of the RB gene have yet been performed on familial RCC.
C
AF
Figure 6 Autoradiogram after PFGE of NotI-digested DNA of normal controls (C) and tumor cells after 4 (A, F) and 6 weeks (S) in culture, from different regions of the tumor (A, F). Use of probes from different regions of the RB gene, Probe p68RS2.0 from the 5' end of the RB gene, was used for the autoradiogram.
30
In a d d i t i o n to the findings d e s c r i b e d above, the t u m o r k a r y o t y p e of our familial RCC s h o w e d a der(16)t(13;?;16) (16pter--~16q24::?::13q14?-~13qter) r e s u l t i n g from an unb a l a n c e d t r a n s l o c a t i o n ; the d e r i v a t i v e 16 was not dicentric. O n e n o r m a l c h r o m o s o m e 13 was missing. Because the RB gene is located in b a n d 13q14.2, this gene m i g h t h a v e b e e n i n v o l v e d . T h e r e f o r e , w e p e r f o r m e d D N A s e q u e n c e polym o r p h i s m and PFGE analysis. W i t h these t e c h n i q u e s , w e d e t e c t e d no large r e a r r a n g e m e n t s or d e l e t i o n s in the RB gene, w h i c h w o u l d h a v e b e e n e x p e c t e d if the t r a n s l o c a t i o n had d i s r u p t e d this locus. Thus, w e did not search for p o i n t m u t a t i o n s in the RB gene. T h i s result s u p p o r t s the idea [20] that in contrast to m a n y o t h e r different t u m o r t y p e s the RB gene m i g h t not be crucial for t u m o r p r o g r e s s i o n or i n i t i a t i o n in this f a m i l i a l RCC. This study was supported in part by NIH Grants No. CA 41183 (A. A. S,) and RO1 CA 49455 (B. R. S.) from the National Cancer Institute. H. J. H. D. received a Research Award from the National Cancer Institute and the European Organization for the Treatment of Cancer. B. R. S. is the recipient of a Faculty Research Award from the American Cancer Society.
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31. Gemmill RM, Coyle-Morris JF, McPeek F, Ware-Uribe LF, Hecht F (1987): Construction of long-range restriction maps in human DNA using pulsed field gel electrophoresis. Gene Anal Tech 4:119-131. 32. Ford GM, Gallie BL, Phillips RA, Becker AJA (19901: Physical map around the retinoblastoma gene. Genomics 6:284-292. 33. Decker HJ, Neumann HP, Walter TA, Sandberg AA (1988): 3p involvement in a renal cell carcinoma in von Hippel-Lindau syndrome. Region of tumor breakpoint clustering on 3p. Cancer Genet Cytogenet 33:59-65. 34. Decker HJ, Gemmill RM, Neumann HP, Walter TA, Sandberg AA (1989): Loss of heterozygosity on 3p in a renal cell carcinoma in von Hippel-Lindau syndrome, Cancer Genet Cytogenet 39:289-293. 35. Seizinger BR, Rouleau GA, Ozelius LJ, Lane AH, Farmer GE, Lamiell JM, Haines J, Yuen JW, Collins D, Majoor Krakauer D, et al. (1988): von Hippel-Lindau disease maps to the region of
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chromosome 3 associated with renal cell carcinoma. Nature 332:268-269. Seizinger BR, Smith DI, Filling Katz MR, Neumann H, Green JS, Choyke PL, Anderson KM, Freiman RN, Klauck SM, Whaley J, Decker HJH, et al. (1991}: Genetic flanking markers refine diagnostic criteria and provide insights into the genetics of Von Hippel Lindau disease. Proc Natl Acad Sci USA 88:2864-2868. Walter TA, Berger CS, Sandberg AA (1989): The cytogenetics of renal tumors. Where do we stand, where do we go? Cancer Genet Cytogenet 43:15-34. Kovacs G. (1990): Application of molecular cytogenetic techniques to the evaluation of renal parenchymal tumors. J Cancer Res Clin Oncol 116:318-323. Kovacs G, Frisch S (1989): Clonal chromosomal abnormalities in tumor cells from patients with sporadic renal cell carcinomas. Cancer Res 49:651-659.
ABSTRACT: In a previously studied f a m i l y with inherited renal cell carcinoma (RCC), RCC was shown to segregate with a constitutional balanced t(3;8)(p14.2;q24.1). In addition, we recently showed that in a RCC tumor from this f a m i l y the constitutional translocation became unbalanced, suggesting a genetic m e c h a n i s m that m a y be associated with the primary genetic events of tumorigenesis. We now report that the RCC tumor cells from this case showed additional cytogenetic alterations, possibly related to tumor progression, which include an additional tumor-specific translocation involving band 14 of chromosome 13. Because this band contains the retinoblastoma (RB) gene, we e x a m i n e d the tumor for aberrations in the RB gene using DNA sequence p o l y m o r p h i s m analysis and pulsed-field gel electrophoresis (PFGE), but did not detect alterations in the RB gene.
INTRODUCTION In 1979, Cohen et al. described renal cell carcinomas (RCC) in 10 adults from three consecutive generations of an Italian-American family [1]. Constitutional karyotypes of 10 of 22 tested family members showed a balanced translocation between chromosomes 3 and 8 which segregated with the RCCs. Wang and Perkins [2] specified the breakpoints of this reciprocal translocation as t(3;8)(p14.2;q24.1). An RCC of this family was studied with a variety of cytogenetic and molecular genetic techniques. Cytogenetic and restriction fragment length polymorphisms (RFLP) studies [3] demonstrated that the only observed tumor cell clone retained the normal chromosome 3, whereas der(8), carrying the distal part of 3p, was lost. Clinical follow-up and some of the genetic data of this case were reported elsewhere [3]. We report a more detailed analysis of genetic alterations in this RCC, which focused on the RB gene. Loss of genetic material from the short arm of chromosome 3 is the most frequently observed nonrandom aberration in both sporadic and familial RCC, suggesting the exis-
From the Molecular Neuro-Oncology Laboratory, Massachu~ setts General Hospital and Harvard Medical School Boston (H.-J. H. D., J. M. W., S. M. K., B. R. S.), Cancer Center of the Southwest Biomedical Research Institute, Scottsdale, Arizona (B. W., A. A. S.), and Department of Ophthalmalogy, Massachusetts Eye and Ear Infirmary, and Harvard Medical School (G. H., D. W. Y.), Boston, Massachusetts. Address reprint requests to Dr. Hans-Joachim H. Decker, Department of Oncology/Hematology, Medical School of the Johannes-Gutenberg-University Mainz, Langenbeck-Str. 1, D-6500 Mainz, Germany. Received December 12, 1991; accepted April 1, 1992.
tence of one or even more tumor suppressor gene(s) in the 3p region. RFLP analyses in sporadic RCC have shown loss of heterozygosity (LOH) at several chromosomal loci other than 3p, but such changes are less frequent than is LOH of 3p. Of particular note is that 15-33% of sporadic RCC have had LOH of genetic loci from 13q [4-6]. LOH of 13q has been found only in advanced cases of RCC, however; therefore, the RB gene, located at 13q14, was suggested to be involved in RCC tumor progression [5l. Loss of RB function has been described in a large variety of tumor types, such as retinoblastomas [7-9], osteosarcomas [9-11], lung cancers [12-14], sarcomas [10, 15, 16], prostate carcinomas [17, 18], and breast cancers [19], but no direct evidence for consistent RB gene involvement in RCC has yet been reported [20]. Because 13q14 may have been involved in an additional unbalanced translocation in the familial RCC in this study, we performed pulsed-field gel electrophoresis (PFGE) and DNA sequence polymorphism analysis of the RB gene to seek aberrations in this tumor suppressor locus. CASE REPORT AND PATHOLOGY
Bilateral partial nephrectomies were performed to remove asymptomatic renal tumors in a 41-year-old white woman. Twelve years later, the patient developed a renal mass in the remnant of the left kidney. She had received no chemotherapy. Total nephrectomy showed a clear cell RCC, various regions of which were provided for genetic studies. MATERIALS AND METHODS Cell Culture and Cytogenetics A piece of tumor was disaggregated [21] with collagenase (final concentration 400 U/ml) and agitated for 4 hours. The
25 © 1992 Elsevier Science Publishing Co., Inc. 655 A v e n u e of the Americas, New York, NY 10010
Cancer Genet Cytogenet 6 3 : 2 5 - 3 1 (1992J 0165-4608/92/$05.00
26 single cells were seeded immediately into flasks, flaskettes, and directly on coverslips. The tumor cells grew attached in RPMI medium, supplemented with 20% fetal bovine serum, L-glutamine, and antibiotics. Nine harvests were performed: the first after 3 days, and the last after 4 weeks. The best results were obtained by a modified synchronization method [22] and the coverslip in situ harvest procedure [23]. GTG- and CBG-banding were performed according to standard procedures [24, 25].
Fluorescence In Situ Hybridization (FISH), DNA Probes, and Cytochemical Detection FISH was done according to the protocol of Pinkel et al. [26] with modifications [27]. Human c~-satellite DNA probes [28] detecting sequences of the centromeric regions of chromosomes 3 and 16 were used: pa3-5 [D3Z1], and pSE 16[D16Z2]. All probes were purchased in the biotin-labeled form from Oncor (Gaithersburg, MD). The intensity of biotin-linked fluorescence was amplified by adding a layer of biotinylated goat anti-avidin antibody to the first layer before adding another layer of fluorescence-avidin DCS (both purchased from Vector Laboratories, Burlingame, CA). The counterstain was propidium iodide in an antifade solution [29].
DNA Sequence Polymorphism (DSP) in RB 1.20 DNA was extracted from 1) cultured RCC cells, 2) normal kidney tissue, and 3) the primary RCC and subsequently PCR-amplified. The primer pair for amplification was derived for the polymorphic sequence RB 1.20 from the 3' end of exon 20: 5'-TGAATGAACAATTGACTAAA-3'(position 431-450) and 5'-CTCTATCTCAGAGTGAGACAATG3'(position 548-569). One of the primers was 32p endlabeled and used in a polymerization reaction with Sequenase (U.S. Biochemical) and all four deoxynucleotides. The reaction products were heat-denatured, electrophoresed on a 6% polyacrylamide gel, and autoradiographed for 12 hours [30].
PFGE and DNA Probes To yield high-molecular-weight DNA, single suspended cells of each sample (tumor cells from different passages, and leukocytes from normal individuals) were imbedded in agarose blocks as previously described [31]. For restriction digestion, Notl was used, because in the long-range physical map around RB the NotI fragment spans 930 (kilobases), including all 200 kb of the retinoblastoma (RB) gene [32]. PFGE was performed in a clamped homogeneous electric field using the LKB pulsaphor system in basic mode. Charged Hybond N + filters were used. The probes for the RB were gifts from Dr. Thaddeus P. Dryja. For hybridization, probes from the 3' end (p123M1.8) and the 5' end (p68RS2.0) of the RB gene were used.
RESULTS Different fractions of the tumor cells in culture exhibited heterogeneous morphologies. Figure 1 shows fractions (A and F) of the tumor. In contrast to the varying morphology,
H.-J. H. Decker et al. cytogenetic, and molecular analysis showed no difference between these separately harvested fractions. Nine harvests were performed after 3-28 days in culture. There was only a single tumor cell clone: 167 cells showed 43,XX,der(3)t(3;8)(p14.2;q24),- 8 , - 1 3 , - 14, der(16)t(13 ;?;16)(16pter--~ 16q24::?::13q14?13qter). Fifteen cells were hypodiploid with random loss. Twelve cells were hypotetraploid; the number of hypotetraploid cells increased with prolonged culture. After 2 weeks of culture, only two cells were detected with the constitutional karyotype 46,XX,t(3;8)(p14.2;q24). These two cells retained the derivative 8 which was lost in the tumor cell clone. In addition, both homologues of chromosomes 13, 14, and 16 were intact in these two cells. Figure 2 shows a partial karyotype with the derivative chromosomes, der(3) and der(16). CBG-banding proved that the derivative chromosome 16 was not dicentric (data not shown). FISH verified the cytogenetic results, showing that the derivative was stemming from chromosome 16 (Fig. 3d) and that two centromeres of chromosome 3 were retained in 85.5-87% of the cells scored. Two signals from the centromere probes of chromosome 16 were observed in 77-90% of cells monitored. Two hundred seventeen (pa35) and 431 (pSE16) nuclei were screened (Fig. 4). DNA sequence polymorphism analysis of RB after polymerase chain reaction (PCR) amplification (Fig. 5) did not show any differences when cultured RCC cells (lane 1), normal kidney tissue (lane 2), and primary RCC (lane 3) of the patient were compared. Two probes from different regions of the RB gene did not detect structural rearrangements after PFGE (Fig. 6) of NotI-digested DNA of controls and tumor cells from morphologically different cultures (compare to Fig. 1A and F) after 4 and 6 weeks (S) in culture.
DISCUSSION The constitutional t(3;8)(p14.2;q24.1) has been clearly associated with development of RCC in 10 members of one family [1, 3]. We describe the genetic alterations observed in the first RCC of this family accessible for cytogenetic studies: the constitutional translocation became unbalanced due to the loss of the derivative (8) carrying the already altered short arm of chromosome 3. Surprisingly, no aberrations could be detected in the normal homologue 3 of our case by cytogenetic and RFLP studies [3]. The retention of both chromosome 3 centromeres, one of the normal homologue and the other of the der(3), was also verified by FISH techniques. These findings may indicate a more complex mechanism of tumorigenesis than simple loss of both copies of a single tumor-suppressor gene. More than one gene on 3p may be involved in the tumorigenesis of RCC; the yon Hippel Lindau gene is one candidate [33-36]. At the time the experiments were performed, no repetitive probes for chromosome 8 centromere were available. Whereas alterations of DNA sequences from 3p appear to be crucial for tumor initiation in sporadic and familial RCC, little is known about genetic alterations leading to tumor progression in RCCs. For sporadic RCC, evidence
27
Figure I Cells from different regions (A, F) of the RCC grown in culture for 2 weeks: Although they show a clearly different morphology, no difference was evident when they were analyzed separately by cytogenetics of PFGE (as compared with Fig. 2).
Figure 2 Selected GTG-banded partial karyotypes showing the structural unbalanced rearrangements observed in three different metaphases: der(3)t(3;8)(p14.2;q24) and der(16)t(13;?;16)(16pter--~16q24::?::13q14?-~13qter).
\ 16q24
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b _i_~ =k
,/
/
13q14?
8q24
c-i- -l3
d e r (3)
U 8
13
der (16)
16
28
d Figure 3 Representative FISH with biotinylated a-satellite probes for the centromeric regions of chromosomes 3[pa3-5] (a and b) and 16[pSE16] (c and d) in metaphases and interphases. Arrow (c) marks the der(16).
F a m i l i a l RCC
29 100%
1{)0% #16
#3
pa 3-5 ID3ZII
pSE 16 IDI6 Z 2l
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I
2
__n 3
>3
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o
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I
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Figure 4 Histogram showing the quantitative analysis of FISH: No significant number of cells with loss of the der(3) was noted in two independent investigations (open and hatched columns).
123 Figure 5 DNA sequence polymorphism analysis after PCR amplification: No differences were noted when cultured 1) RCC cells 2) normal kidney tissue, and 3) and the primary RCC of the patient were compared.
shows i n v o l v e m e n t of other c h r o m o s o m a l loci than 3p, but all of these are less frequent than 3p loss [37, 38]. In our case, in a d d i t i o n to c h r o m o s o m e 3 and 8 alterations, one c h r o m o s o m e 14 was lost in all tumor cells. Although m o n o s o m y 14 is c o m m o n in sporadic and familial RCC (30% in - 3 0 0 RCCs and RCC cell lines reported so far), the significance of this is not known, but it could be related to t u m o r progression [39]. In three previous studies [4-6], LOH of DNA fragments from 13q was observed in a d v a n c e d sporadic RCC. This led to the proposal that the RB gene might be altered during tumor progression in RCC [5], but for sporadic RCC, the RB gene was recently shown to be infrequently involved by the means of Southern, Northern, and Western blotting and i m m u n o s t a i n i n g [20]. No m o l e c u l a r studies of the RB gene have yet been performed on familial RCC.
C
AF
Figure 6 Autoradiogram after PFGE of NotI-digested DNA of normal controls (C) and tumor cells after 4 (A, F) and 6 weeks (S) in culture, from different regions of the tumor (A, F). Use of probes from different regions of the RB gene, Probe p68RS2.0 from the 5' end of the RB gene, was used for the autoradiogram.
30
In a d d i t i o n to the findings d e s c r i b e d above, the t u m o r k a r y o t y p e of our familial RCC s h o w e d a der(16)t(13;?;16) (16pter--~16q24::?::13q14?-~13qter) r e s u l t i n g from an unb a l a n c e d t r a n s l o c a t i o n ; the d e r i v a t i v e 16 was not dicentric. O n e n o r m a l c h r o m o s o m e 13 was missing. Because the RB gene is located in b a n d 13q14.2, this gene m i g h t h a v e b e e n i n v o l v e d . T h e r e f o r e , w e p e r f o r m e d D N A s e q u e n c e polym o r p h i s m and PFGE analysis. W i t h these t e c h n i q u e s , w e d e t e c t e d no large r e a r r a n g e m e n t s or d e l e t i o n s in the RB gene, w h i c h w o u l d h a v e b e e n e x p e c t e d if the t r a n s l o c a t i o n had d i s r u p t e d this locus. Thus, w e did not search for p o i n t m u t a t i o n s in the RB gene. T h i s result s u p p o r t s the idea [20] that in contrast to m a n y o t h e r different t u m o r t y p e s the RB gene m i g h t not be crucial for t u m o r p r o g r e s s i o n or i n i t i a t i o n in this f a m i l i a l RCC. This study was supported in part by NIH Grants No. CA 41183 (A. A. S,) and RO1 CA 49455 (B. R. S.) from the National Cancer Institute. H. J. H. D. received a Research Award from the National Cancer Institute and the European Organization for the Treatment of Cancer. B. R. S. is the recipient of a Faculty Research Award from the American Cancer Society.
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