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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 5 (March 1), 1998:
pp. 1680-1687
p16/INK4a and p15/INK4b Gene Methylation and Absence of p16/INK4a
mRNA and Protein Expression in Burkitt's Lymphoma
By
Ulf Klangby,
Ismail Okan,
Kristinn P. Magnusson,
Martin Wendland,
Peter Lind, and
Klas G. Wiman
From the Microbiology & Tumor Biology Center, Karolinska Institute;
and Pharmacia & Upjohn, Stockholm, Sweden.
 |
ABSTRACT |
The fact that the p16/INK4a and p15/INK4b genes are frequently
inactivated in human malignancies and that p16/INK4a null mice spontaneously develop B-cell lymphomas prompted us to examine the
status of both genes in Burkitt's Lymphoma (BL). We found a low
frequency of p16/INK4a and p15/INK4b deletions and mutations in BL cell
lines and biopsies. However, p16/INK4a exon 1 was methylated in 17 out
of 19 BL lines (89.5%) and in 8 out of 19 BL biopsies (42%) analyzed.
p15/INK4b Exon 1 was also methylated, although at a lower frequency.
p16/INK4a mRNA was readily detected in BL lines carrying unmethylated
p16/INK4a, but not in those carrying methylated p16/INK4a. No p16/INK4a
protein was detected in any of the BL lines and biopsies examined. In
contrast, only one out of seven lymphoblastoid cell lines (LCLs)
examined was methylated in p16/INK4a exon 1, and three out of the six
LCLs with unmethylated p16/INK4a expressed detectable levels of
p16/INK4a protein. Thus, the frequent p16/INK4a methylation in BL lines
correlates with downregulation of p16/INK4a expression, suggesting that
exon 1 methylation is responsible for silencing the p16/INK4a gene in BL.
| |
INTRODUCTION |
CELL CYCLE progression is regulated by
complexes formed between cyclins and cyclin-dependent kinases (CDKs).
The kinase activity of CDK4 and CDK6 is activated on association with
D-type cyclins in the G1 phase of the cell cycle.1 The
cyclin D-CDK 4/6 complexes can phosphorylate the retinoblastoma (RB)
protein in vitro, suggesting that cyclin D-CDK4/6 complexes positively
regulate progression through the G1 phase of the cell cycle by
phosphorylating RB, thereby eliminating the cell cycle block imposed by
RB.2 The activity of cyclin D-CDK4/6 complexes is blocked
by cyclin-dependent kinase inhibitors (CDIs).3 The
p16/INK4a and p15/INK4b proteins, both members of the INK4 family of
CDIs, bind CDK4/6 and block their complexing with D type
cyclins.4-6 This prevents RB phosphorylation and
progression into S phase, thus arresting the cell in the G1 phase.
Homozygous deletion of the p16/INK4a gene and the p15/INK4b gene that
maps in tandem with p16/INK4a on chromosome 9p21 was first observed in
many human tumor cell lines7,8 and has subsequently been
found in a wide variety of human primary tumors, for instance gliomas,
acute lymphoblastic leukemias, and prostate and bladder
carcinomas.1 In addition, point mutations and small deletions in the p16/INK4a gene occur in certain tumor types, including
pancreatic adenocarcinomas and esophageal and biliary tract carcinomas,
and are associated with familial melanoma.1 Methylation of
the CpG islands within the p16/INK4a and p15/INK4b genes, detected in
breast and colon carcinomas, is yet another mechanism for silencing
these genes.9-13 All these findings indicate that at least
p16/INK4a is an important tumor suppressor gene.
A second p16/INK4a transcript that initiates from a more 5 promoter
encodes an entirely different protein, p16 or p19ARF, caused by
splicing of the alternative exon 1 to the common exon 2, initiation
of translation from an AUG within exon 1, and usage of an
alternative reading frame in exon 2.14-17 The p16/p19ARF protein has been shown to induce both G1 and G2 arrest in NIH3T3 cells.17 Although at least some of the point mutations in
p16/INK4a found in human tumors affect both the regular p16 protein and p16/p19ARF, the role of the latter in tumorigenesis remains unclear.
Because inactivation of p16/INK4a and possibly p15/INK4b is a common
mechanism for disruption of cell cycle control in human tumors, and
because p16/INK4a null mice develop B-cell lymphomas with a high
incidence,18 we decided to study the involvement of these
genes in Burkitt's lymphoma (BL). Multiple factors contribute to the
origin of this tumor. All BL carry chromosomal translocations that
activate the c-myc proto-oncogene by juxtaposition to one of the Ig
heavy and light chain loci. Most endemic BL and some sporadic BL carry
Epstein-Barr virus (EBV).19 In addition, the p53 gene is
mutated in a large fraction of BL biopsies and lines.20-22 Non-neoplastic EBV-immortalized lymphoid cell lines (LCLs), on the
other hand, lack Ig-myc translocations and express wild type p53. Here
we show that the p16/INK4a and p15/INK4b loci are frequently methylated
in BL but not in LCL, and that p16/INK4a methylation correlates with
silencing of the p16/INK4a gene in BL lines.
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MATERIALS AND METHODS |
Tumor biopsies and cell lines.
Twenty-one primary BL biopsies from endemic areas in Africa were
provided by George Klein, Microbiology & Tumor Biology Center, Karolinska Institute. More than 90% of the cells were tumor cells in
these biopsies. Forty-three BL cell lines and 12 LCLs were also
included (Table 1). Cells were grown in
RPMI 1640 or Iscove's modified Dulbecco's medium (IMDM) supplemented
with antibiotics and 10% fetal bovine serum (FBS). Normal human B
cells were obtained from healthy donors using CD19-loaded magnetic
beads (Dynal, Oslo, Norway).
Preparation of genomic DNA and polymerase chain reaction (PCR)
amplification.
Genomic DNA was prepared as described.23 PCR was performed
using 10 pmol of each primer, 250 µmol/L of dNTP mix, 5% dimethyl sulfoxide (DMSO), 1 U Taq Polymerase (Pharmacia, Uppsala, Sweden, or
Perkin Elmer, Norwalk, CT), standard Taq buffer, and 50 to 100 ng of
genomic DNA. p16/INK4a Exon 2 was amplified using the 42F and 551R
primers, and p15/INK4b exon 2 was amplified using the 89F and 50R
primers.7 PCR was performed for a total of 35 cycles using
an annealing temperature of 58°C (p16/INK4a) or 56.5°C (p15/INK4b).
PCR products were analyzed on EtBr-stained 1.2% agarose gels. The
quality of the genomic DNA was confirmed by parallel PCR amplification
of a GAPDH gene exon 8 fragment, using the same conditions as for PCR
amplification of p15/INK4b, and the primers 5-CCCTCCGGGAAACTGTGGCGT-3
(GAPDHE8F) and 5-ATGCCAGCCCCAGCGTCAAAG-3 (GAPDHE8R). All PCR
reactions were repeated at least twice.
Southern blot analysis.
Southern blotting was performed using standard
procedures.23 A 126-bp fragment corresponding to nt 107-233 of p16/INK4a exon 2 was used as a probe to detect both p16/INK4a and
p15/INK4b.7 The probe was radioactively labeled using
Megaprime DNA labeling system (Amersham, Buckinghamshire, UK) following
the manufacturer's instructions. Ten micrograms of
EcoRI-digested genomic DNA from each sample was separated on
1% agarose gels and transferred onto Hybond N+ filters
(Amersham). Hybridizing bands were visualized by
autoradiography or Phospholmager.
DNA sequencing.
Sequencing reactions were performed using the Taq DyeDeoxy Terminator
Cycle Sequencing Kit (Applied Biosystems, CT) according to the
manufacturer's instructions. All sequencing reactions were performed
on pooled PCR samples from three different reactions. The primers used
for sequencing of p16/INK4a exon 1 were 2F and 1108R.7 The
primers used for sequencing of exon 2 of p16/INK4a and p15/INK4b were
42F and 551R, and 89F and 50R, respectively.
Reverse transcription (RT)-PCR.
cDNA was synthesized from total RNA using M-MLV Reverse Transcriptase
(GIBCO-BRL, Gaithersburg MD) according to the manufacturer's instructions. RT-PCR was performed using 10 pmole of each primer, 250 µmol/L of dNTP mix, 5% (p16 and p16) or 3% (p15) DMSO, and 1 U
of Taq Polymerase (Pharmacia) and standard Taq buffer. PCR was
performed for a total of 35 cycles using an annealing temperature of
61°C and the primers 5-ACTAGATCTTCGCACGAGGCAGCATGG-3 (p16E1F) and
5-GTTGTGGCGGGGGCAGTTGT-3 (p16E3R) for p16 cDNA;
5-AAGGATCCATGGTGCGCAGGTTCTTGG-3 (p16BF) and p16E3R (see above) for
p16 cDNA; and 5-GTTTACGGCCAACGGTGGA-3 (p15E1F) and
5-GCAGAATTCATCGAATTAGGTGGGTGG-3 (p16E2R) for p15 cDNA. Ten
microliters from each reaction was run on 1% agarose gels and then
transferred onto Hybond N+ membranes (Amersham). Membranes were probed
with 32P-dCTP-labeled probes corresponding to exon 1 of the respective
genes. As control, GAPDH cDNA was amplified by RT-PCR using the primers
5-TGCCTCCTGCACCACCAACTG-3 (GAPDHE7F) and GAPDHE8R (see above) for
each sample tested. Hybridizing bands were visualized by PhosphoImager.
Northern blot analysis.
Northern blotting was performed as described.23 A 175-bp
BamHI-BgII p16/INK4a cDNA fragment corresponding to the entire coding region of exon 1 and the first 20 bp of the coding region of
exon 2 was used as probe. Twenty micrograms of total RNA was loaded in
each lane. Hybridizing bands were visualized by PhosphoImager.
Western blot analysis.
Detection of p16 protein by immunoblotting was performed as
outlined.24 Cell extracts were prepared from subconfluent
cultures by resuspending 1 × 107 cells in 100 µL of
Laemmli sample buffer. Total protein from each sample (50 to 150 µg)
was separated by sodium dodecyl sulfate (SDS) polyacrylamide gel
electrophoresis and transferred onto nitrocellulose. p16 was detected
with the rabbit polyclonal antiserum 15126E and/or the mouse
monoclonal antibody 13251A (Pharmingen, CA), followed by ECL (Amersham)
according to the manufacturer's instructions. Actin was detected with
the mouse antibody N350 (Amersham).
Methylation assay.
Genomic DNA from primary tumors and cell lines was digested overnight
with SacII (Pharmacia), HpaII (MBI Fermentas, Vilnius, Lithuania; GIBCO BRL; and Pharmacia) or MspI (New England
Biolabs, Beverly, MA), using 20 U enzyme/µg DNA. PCR was performed
using 10 pmol of each primer, 250 µmol/L of dNTP mix, 5% DMSO, 1 U
Taq Polymerase (Pharmacia) standard Taq buffer, and 125 ng of genomic DNA. PCR was performed for a total of 21 cycles using an annealing temperature of 59°C and the primers 5-ATGGAGCCTTCGGCTGAC-3
(p16metF), 2F and 1108R for p16/INK4a exon 1, and
5-TTTCCCAGAAGCAATCCA-3 (p15metF) and 5-TGTCGCACCTTCTCCACT-3 (p15
metR) for p15/INK4b exon 1. Ten microliters from each reaction was run
on 1% agarose gels and transferred onto nylon membranes (Hybond N+;
Amersham). Membranes were probed with 32P-dCTP-labeled probes
corresponding to part of exon 1 of the respective genes (Fig
1). Hybridizing bands were
visualized by PhosphoImager. All restriction digestions, PCR reactions,
and transfers were repeated at least twice.
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| Fig 1.
Strategy for detection of gene methylation using PCR
followed by hybridization with an internal probe. Primers used are
indicated by the arrows. Filled lines represents probes. Cleavage sites for HpaII/Msp I (H) and SacII (S) are
indicated. The gene map is not drawn to scale.
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RESULTS |
Analysis of p16/INK4a and p15/INK4b by PCR and Southern blotting.
Forty-three BL lines and 12 LCLs were screened for homozygous deletion
of p16/INK4a and p15/INK4b by PCR, using primers surrounding exon
2.7 All 12 LCLs and 40 out of 43 BL lines retained exon 2 of both p16/INK4a and p15/INK4b. Three BL lines (BL2, BL28, and Ew36;
7%) showed homozygous deletion of exon 2 of p16/INK4a, and two lines
(BL2 and BL28; 4.6%) also had homozygous deletion of exon 2 of
p15/INK4b (Fig 2, lanes 3, 5, and 7; Table
1). These results were confirmed by Southern blotting (data not shown). Iarc139, an LCL derived from the same patient as BL28, retained both
p16/INK4a and p15/INK4b, showing that homozygous deletion of p16/INK4a
and p15/INK4b is a somatic rather than a constitutional change.
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| Fig 2.
PCR amplification of exon 2 of p16/INK4a (top) and
p15/INK4b (middle). BL2 (lane 3) and BL28 (lane 5) had homozygous
deletion of exon 2 of both p16/INK4a and p15/INK4b, whereas Ew36 (lane 7) had homozygous deletion of p16/INK4a exon 2 but retained p15/INK4b exon 2. The expected p16/INK4a and p15/INK4b PCR products were obtained
from genomic DNA from all primary BL biopsies indicated in Table 1. Two
examples, MK and TO, are shown (lanes 1 and 2). Lanes 3 to 10, BL
lines; lane 11, LCL. DNA integrity was confirmed using primers specific
for GAPDH exon 8 (bottom).
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The status of the p16/INK4a and p15/INK4b genes was also examined in 21 primary BL biopsies. PCR analysis showed that all BL biopsies retained
exon 2 of both genes. Representative examples of this analysis are
shown in Fig 2 (lanes 1 and 2). These results were confirmed by
Southern blotting (data not shown).
Analysis of p16/INK4a and p15/INK4b mRNA and protein expression.
p16/INK4a mRNA levels in the BL lines were examined by Northern
blotting and RT-PCR. As expected, no p16/INK4a mRNA was detected in the
BL lines that carried homozygous p16/INK4a deletion. Surprisingly, however, Northern blot analysis did not reveal any p16/INK4a mRNA in a
majority of the BL lines that had retained the p16/INK4a gene. Only 1 out of 20 BL lines (Seraphine) and 2 out of 6 LCLs analyzed (Iarc139
and Iarc174) expressed levels of p16/INK4a mRNA detectable by Northern
blotting (data not shown). Using the more sensitive RT-PCR technique
followed by hybridization with an internal probe, p16/INK4a mRNA was
detected in Seraphine and BL18, and in the LCLs Cherry, Iarc139,
Iarc174, and Nad20 (Fig 3; Table 1). After
long exposure, low levels of p16/INK4a mRNA were also found in the BL
lines BL60 and CA46 (not shown). The alternative p16/INK4a transcript,
p16, was expressed in all BL lines with intact p16/INK4a gene (Fig
3). Using RT-PCR, low levels of p15/INK4b mRNA were detected in all BL
lines that carried an intact p15/INK4b gene, except BL18. p15/INK4b
mRNA was also detected in all six LCLs tested, and in normal B cells
(Fig 3; Table 1).
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| Fig 3.
Detection of p16/INK4a, p16/INK4a, and p15/INK4b mRNA
by RT-PCR and hybridization with an internal probe. After
electrophoresis, PCR products were transferred to nylon filters and
hybridized with probes corresponding to exon 1 of p16/INK4a,
p16/INK4a, p15/INK4b, and rat GAPDH cDNA. Hybridizing bands were
visualized by Phospholmager.
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Western blotting revealed high levels of p16 protein in the Saos-2 and
HeLa cell lines used as positive controls. However, no p16 protein was
observed in any of the 4 BL biopsies and 27 BL lines examined (Fig
4; Table 1). Among the LCLs, Iarc139
expressed comparatively high levels of p16 protein (Fig 4), and Nad20
and Iarc174 expressed low levels detectable after long exposure (Fig 4
and data not shown). The remaining LCLs and normal human B cells did
not express detectable levels of p16 protein (Table 1).
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| Fig 4.
Western blot analysis of p16 expression in BL biopsies
(left panel), cell lines, and LCLs (right panel). HeLa cells were used as positive control. An actin antibody was used as an internal control
to confirm protein integrity.
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Methylation of the p16/INK4a and p15/INK4b genes.
To determine whether the lack of p16/INK4a expression in the BL lines
was caused by methylation of the CpG island in p16/INK4a exon 1, we
used a previously described PCR strategy9 (Fig 1). Genomic
DNA from primary BL biopsies, BL lines, LCLs, and normal human B cells
were digested with the methylation-sensitive restriction enzymes
SacII or HpaII, or the methylation-insensitive enzyme Msp I, an isoschizomer of HpaII. Digested and intact
DNA were subsequently analyzed by PCR followed by hybridization with an internal probe (Fig 1). Representative examples of this analysis using
the 3 of the two forward primers are shown in Fig
5. Eight out of 19 primary BL biopsies
(42%) examined were methylated at the 3 HpaII site.
Methylation of both HpaII sites, as shown by the appearance of
a PCR product following HpaII cleavage when the 5 of the two
forward primers was used, was not observed in any of the primary BL.
Seventeen out of 19 BL lines (89.5%) were methylated at the 3
HpaII site. The 5 HpaII site and the SacII site were less frequently methylated than the 3 HpaII site in the BL lines (not shown). Only two BL lines, Seraphine and BL18, both
of which expressed p16/INK4a mRNA, were unmethylated in the region
examined (Fig 5; Table 1). Thus, p16/INK4a exon 1 methylation occurs
with high frequency in BL. In contrast, six out of seven LCLs and
normal human B cells were unmethylated in this region (Fig 5; Table 1).
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| Fig 5.
Methylation of exon 1 of p15/INK4b (bottom panel) and
p16/INK4a (top panel) in representative BL biopsies, BL lines, LCLs, and normal B cells. Genomic DNA was digested with HpaII (H),
Msp I (M), or left undigested (U), and analyzed by PCR followed
by hybridization with an internal probe, as shown in Fig 1.
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Methylation of the p15/INK4b gene was studied using the same approach,
except that only one forward primer was used (Fig 1). Twelve out of 20 BL lines (60%) and 5 out of 19 BL biopsies (26%) were methylated at
all four HpaII sites in p15/INK4b exon 1. p15/INK4b Methylation
was not found in normal B cells nor in six out of seven LCLs
investigated (Fig 5; Table 1).
DNA sequence analysis of p16/INK4a and p15/INK4b.
DNA sequencing of exon 2 of p16/INK4a and p15/INK4b did not reveal any
mutations in the 7 BL biopsies analyzed (Table 1). In addition, exons 1 and 2 of p16/INK4a in the BL lines Seraphine, BL18 (both carrying
unmethylated p16/INK4a) and Akuba (carrying methylated p16/INK4a) were
sequenced. This analysis showed a G to T substitution at nucleotide 298 in exon 2 of the p16/INK4a gene in Seraphine, resulting in an Ala to
Ser substitution at codon 100. No mutations were found in exons 1 and 2 of p16/INK4a in BL18 and Akuba.
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DISCUSSION |
We found that only a minor fraction of the BL lines had homozygous
deletion of the p16/INK4a and p15/INK4b genes, confirming studies by
Stranks et al25 and Herman et al.26 Thus, BL
differs in this respect from for instance acute lymphoblastic
leukemias, which often carry homozygous deletion of
p16/INK4a.27 Moreover, DNA sequencing did not reveal any
small deletions or point mutations in exon 2 of p16/INK4a and p15/INK4b
in the BL biopsies examined, nor in p16/INK4a exon 1 and 2 in the BL
lines BL18 and Akuba. These results thus indicate that inactivation of
the p16/INK4a and p15/INK4b genes by structural alterations is
infrequent in BL.
Although most BL lines had retained the p16/INK4a gene, only 4 BL lines
expressed detectable levels of p16/INK4a mRNA as determined by RT-PCR
followed by hybridization with an internal probe. Because methylation
of the p16/INK4a and p15/INK4b genes has been associated with
downregulation of their expression in several tumor
types,9-13 we asked whether p16/INK4a and p15/INK4b gene
methylation occurred in our BL lines and biopsies. We found methylation
of exon 1 of the p16/INK4a gene in a majority (89.5%) of the BL lines
and in a large fraction (42%) of the primary BL biopsies. A similar
frequency was reported in a study of eight primary BL
tumors.26 We did not observe p16/INK4a gene methylation in
any of the LCLs examined except Iarc171, nor in normal human B cells,
consistent with the possibility that methylation is a BL-associated
phenomenon responsible for silencing the p16/INK4a gene. In support of
this idea, methylation of p16/INK4a exon 1 showed an inverse
correlation with p16 mRNA expression in the BL lines and LCLs. The only
two BL lines that were not methylated in p16/INK4a exon 1, BL18 and
Seraphine, expressed p16/INK4a mRNA levels detectable by RT-PCR (Fig
3). Two other BL, CA46 and BL60, both carrying a methylated p16/INK4a
gene, also expressed p16/INK4a mRNA. However, the levels of p16/INK4a mRNA expressed by CA46 and BL60 were very low and could only be detected after long exposure of the RT-PCR blots. It is possible that
complete silencing of the p16/INK4a gene requires methylation of
additional sites outside the region studied here, and that these sites
are not methylated in CA46 and BL60. At least four of the LCLs that
carried unmethylated p16/INK4a expressed p16/INK4a mRNA as determined
by Northern analysis and/or RT-PCR; the only LCL that carried
methylated p16/INK4a (Iarc171) did not express p16/INK4a mRNA and
protein. Thus, p16/INK4a methylation occurred with high frequency in
the BL biopsies and lines but not in the LCLs, and was associated with
silencing of this gene in the BL lines, suggesting the possibility that
p16/INK4a inactivation through this mechanism is involved in the
development of BL. The observation that the frequency of p16/INK4a gene
methylation is higher in BL lines than primary tumors indicates a
selection for p16/INK4a gene inactivation also during in vitro culture.
A corresponding discrepancy between the mutation rate in primary BL
biopsies and BL lines exists for the p53 gene.21,28
Exon 1 of p15/INK4b was also methylated in the BL lines and primary
tumors, although at a lower frequency than p16/INK4a. Many BL lines
with methylated p15/INK4b expressed p15/INK4b mRNA, as shown by RT-PCR
followed by hybridization with an internal probe (Table 1). This is
consistent with the observation that p15/INK4b gene methylation does
not necessarily lead to complete silencing of the gene.9
However, we cannot exclude the possibility that a fraction of each
sample contains cells with unmethylated p15/INK4b that account for the
p15/INK4b mRNA detected.
DNA sequencing revealed a point mutation in the coding sequence of
p16/INK4a in the BL line Seraphine. This gives rise to an Ala to Ser
substitution at residue 100 in the p16 protein. We have no experimental
data regarding the consequences of this mutation for p16 function, but
the fact that it occurred in one of the two BL lines carrying an
unmethylated p16/INK4a gene is consistent with the idea that point
mutation represents an alternative, uncommon mechanism for p16/INK4a
inactivation in BL.
The cytogenetic hallmark of BL is the chromosomal translocations that
activate the c-myc proto-oncogene. The c-myc protein has been shown to
activate cdc25A, a gene encoding a CDK-activating phosphatase expressed
in the early G1 phase of the cell cycle.29 CDK4 and CDK6,
two kinases that form complexes with D cyclins in the G1 phase and
phosphorylate the RB protein, are putative substrates of cdc25A. Thus,
one consequence of constitutive c-myc activation in BL may be
disruption of normal G1 cell cycle control through increased RB
phosphorylation. Nevertheless, our observation that p16/INK4a is often
methylated in BL indicates that p16/INK4a inactivation may provide a
selective growth advantage even in the presence of constitutively
active c-myc. The reason may be that activation of the cdc25A
phosphatase would not result in CDK4 or CDK6 activation if p16/INK4a is
expressed. p16/INK4a could thus be considered dominant over cdc25A in
this respect. This notion is supported by the observation that
p16/INK4a can block transformation of primary rat embryo fibroblasts by
c-myc and mutant ras.30
A large fraction of BL also carry p53 mutations. Because c-myc is known
to induce apoptosis in a p53-dependent manner,31 it is
possible that constitutive c-myc expression would lead to a selection
for p53 mutations that would prevent p53-induced apoptosis. Moreover,
functional RB has been shown to inhibit p53-dependent apoptosis.32 Loss of p16/INK4a, resulting in functional RB
inactivation through CDK4/6-mediated phosphorylation, may therefore
promote p53-dependent apoptosis as well, and further increase the
selection for p53 mutation.
EBV is another factor involved in the genesis of BL. All BL lines that
had homozygous deletion of p15/INK4b and/or p16/INK4a, namely
BL2, BL28, and Ew36, are EBV negative. However, the BL lines and
biopsies that carried p16/INK4a methylation included both EBV positive
and EBV negative BL (for example the EBV positive Daudi, Namalwa, and
WW2-BL, and the EBV negative BL41, DG75, and Ramos).22 This
suggests that EBV does not reduce the selection for p16/INK4a gene
inactivation during BL development. The situation may be different in
LCLs. In contrast to primary BL and phenotypically representative BL
lines, LCLs express the EBV-encoded nuclear antigens EBNA-2-6 and the
membrane proteins LMP-1, -2A, and -2B, several of which are important
for transformation of B cells.19 Conceivably, these
transformation-associated EBV-encoded proteins may allow unlimited
growth despite the expression of p16/INK4a in LCLs.
We detected the alternative p16/INK4a transcript, p16, by RT-PCR
and/or Northern blotting in all BL lines carrying an intact p16/INK4a gene (Fig 3; data not shown). This is in agreement with the
notion that methylation of p16/INK4a exon 1 does not silence p16
transcription, and that p16 is usually not targeted during BL
development. The BL with homozygous deletion of p16/INK4a, which knocks
out both the regular and the p16 transcript, are obvious exceptions.
Moreover, the G to T base substitution in exon 2 of p16/INK4a in
Seraphine that replaces Ala with Ser in the regular p16 protein also
affects the p16 protein, causing a Gly to Val change at codon 114. We cannot exclude that p16/INK4a exon 1 or other regions of the
p16/INK4a gene that were not analyzed have suffered point mutations or
minor deletions that inactivate p16 in other BL lines studied here.
Nevertheless, the frequent p16/INK4a methylation and the relatively
infrequent homozygous deletion of this gene as shown in this study
indicate a selection for inactivation of the regular p16INK4a protein
but not the p16 protein in BL.
| |
FOOTNOTES |
Submitted March 24, 1997;
accepted October 14, 1997.
Supported by grants from the Gustaf V Jubilee Fund and the Swedish
Cancer Society (Cancerfonden). U.K. was supported by Pharmacia & Upjohn.
Address reprint requests to Klas G. Wiman, Microbiology & Tumor Biology
Center, Karolinska Institute, Doktorsringen 13, S-171 77 Stockholm,
Sweden.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank George Klein, Karolinska Institute, for providing BL lines and
biopsies and LCLs, and Alexander Kamb, Myriad Genetics, for the
p16/INK4a cDNA.
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Abstract
Introduction
Abstract