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From the Departments of Genetics*
and
Pathology,
"Virgen de la Salud" Hospital,
Toledo, and Department of Genetics,
Fundación J. Díaz, Madrid, Spain
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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p16/INK4A is frequently inactivated by biallelic deletion, although hemizygous deletion associated with mutation or allelic rearrangement has also been described in different types of malignancies. These genetic alterations at the 9p21 region have been found in a high percentage of tumor cell lines (70 to 80%),6-8 whereas in primary human tumors they have been identified with a slightly lower frequency (10 to 70% of cases).9-13 Previous studies in non-Hodgkin's lymphomas (NHL) have detected deletions and/or mutations at the p16/INK4A gene in a relatively low proportion of cases (0 to 14%); this proportion was higher in T-acute lymphoblastic leukemia.14-18
Inactivation of the p16/INK4A gene has also been described as arising due to de novo methylation at the 5'-CpG island, leading to transcriptional blockage of full-length p16/INK4A,19 while permitting the expression of a shorter transcript with a different exon 1 (p16-ß).20
The aim of this study was to determine whether or not p16/INK4A inactivation is involved in the development of NHLs. Because p16/INK4A inactivation has already been found to be the final consequence of several genetic alterations, we decided to complement molecular study with immunohistochemical analysis of p16/INK4A expression, as a majority of the p16/INK4A gene alterations characterized to date eventually give rise to the absence or reduction of the p16/INK4A protein. To this end, we selected a series of NHLs with various histological types, also including selected specimens from cases showing tumor progression from low- to high-grade lymphoma. Reactive lymphoid tissue samples (tonsil and lymph node) were included to define references of p16/INK4A staining in nontumor lymphoid cells. In vitro studies on resting and mitogenically stimulated peripheral blood lymphocytes (PBLs) were also performed to characterize changes in p16/INK4A protein expression along the cell cycle.
To determine the underlying genetic mechanism associated with loss of expression, a group of 26 cases was analyzed for loss of heterozygosity (LOH), mutational spectrum, and de novo methylation pattern.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Normal peripheral blood was obtained by venipuncture from voluntary healthy donors. PBLs were isolated by Histopaque (Sigma Diagnostics, St. Louis, MO) density gradient centrifugation and washed in RPMI 1640. Cells were kept at 37°C in a 5% CO2 humidified incubator in cell culture flasks at 2 x 106 cells/ml of RPMI 1640 supplemented with 10% fetal calf serum, 2 mmol/L L-glutamine, and 2% phytohemagglutinin (PHA) (Life Technologies, Inc., Grand Island, NY). Aliquots of the activated cells were harvested every 24 hours and prepared for analysis.
Control cell lines included in the study were Molt-4 (a T-acute lymphoblastic leukemia cell line defective in the p16/INK4A gene, with deletion of one allele and rearrangement of the other)21 and Saos-2 (an osteosarcoma cell line defective in p53 and RB genes). These cell lines were obtained from the American Type Culture Collection (Manassas, VA).
Western Blot
Aliquots from cell lines were collected at confluence. Aliquots from Molt-4 and Saos-2 cell lines, PBLs, and PHA-stimulated PBLs were washed twice with cold phosphate-buffered saline (PBS) before protein extraction procedure. Protein was extracted with a triple-detergent lysis buffer (50 mmol/L Tris-Cl (pH 8.0), 150 mmol/L NaCl, 0.02% sodium azide, 0.1% sodium dodecyl sulfate, 1% Nonidet P-40, 0.5% sodium deoxycholate, 100 µg/ml phenylmethylsulfonyl fluoride, and 50 µg/ml aprotinin) for 30 minutes at 4°C. Extracts were cleared by centrifugation. Fifty µg of extracted protein was resolved by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose-ECL (Amersham, Poole, UK) according to the manufacturer's instructions. The blots were blocked overnight with 5% bovine serum albumin in PBS at 37°C. p16/INK4A protein was detected by using the monoclonal antibody DCS-50.1 (Calbiochem, Oncogene Research Products, La Jolla, CA), followed by incubation with goat anti-mouse antibody coupled to horseradish peroxidase (Amersham). Blots were developed using the enhanced chemiluminescence ECL detection kit (Amersham).
Flow Cytometry
The expression of p16/INK4A was also studied by flow cytometry (FCM) analysis of PHA-activated lymphocytes. To make it possible to detect this protein by immunofluorescence, fixation/permeabilization for optimal immunodetection was performed with paraformaldehyde/Triton X-100, according to the method of Aiello et al.22 The monoclonal antibody used was the same as in the Western blot (see above). An isotype-matched negative control was used to estimate the quantity of nonspecific binding.
Before labeling, the cells were incubated for 30 minutes with 20% goat serum. They were then incubated for 30 minutes at 4°C with antibodies, followed by two consecutive washes in 0.1% bovine serum albumin diluted in PBS. For p16/INK4A detection, cells were incubated once again (30 minutes at 4°C) with goat anti-mouse monoclonal antibody (F(ab)'2 fragments) conjugated with fluorescein isothiocyanate (Dakopatts, Glostrup, Denmark). After two more washes, the cells were analyzed.
Parallel samples of PHA-activated lymphocytes were prepared for FCM analysis of their nuclear DNA content using the CycleTEST DNA Reagent Kit (Becton Dickinson Immunocytometry Systems, Mountain View, CA).
Double staining for p16/INK4A and DNA was performed in PHA-activated lymphocytes that had previously been fixed, permeabilized, and indirectly labeled for p16/INK4A using a fluorescein isothiocyanate-tagged secondary antibody. The lymphocytes were then incubated (30 minutes at room temperature) with 100 µl of RNase (2 mg/ml) and 200 µl of propidium iodide (2 mg/ml).
FCM was performed using a FACSORT instrument (Becton Dickinson, San Jose, CA), equipped with a double discrimination module. Cell cycle analysis was carried out using the CELLFIT software package for data acquisition and analysis. Protein analysis was undertaken using the LYSIS II software program.
In Vivo Studies
Formalin-fixed paraffin embedded-tissue sections from 153 NHLs samples, 4 reactive tonsils, and 4 cases of lymphadenitis were selected from the routine files of the Pathology Department of the "Virgen de la Salud" Hospital, Toledo, Spain.
Lymphomas were classified according to the REAL classification,23 including 9 cases of B cell chronic lymphocytic leukemia, 6 cases of splenic marginal zone lymphoma, 8 cases of mantle cell lymphoma , 8 cases of low-grade (small cell) mucosa-associated lymphoid tissue (MALT) lymphoma, 10 cases of follicular lymphoma (FL), 23 cases of diffuse large B cell lymphoma of nodal origin (nodal DLBCL), 20 cases of DLBCL arising in mucosa (mucosa DLBCL), 14 cases of Burkitt's lymphoma, 12 cases of peripheral T-cell lymphoma, and 2 cases of anaplastic large cell lymphoma.
For practical purposes, we denominated those composed mainly of the small cell component as "low-grade MALT lymphoma," whereas large cell tumors located in gastric or other mucosa without an adjacent low-grade component were termed "mucosa DLBCL."
To address the possible role of p16/INK4A in tumor progression, we included samples of MALT lymphoma and FL showing aggressive transformation. Thus, samples from 37 cases of MALT lymphoma including simultaneously separate areas of small cells (low grade) and large cells (high grade) were included. These composite tumors in which both components (separate areas of small and large cells) could be discerned were denominated "MALT + DLBCL." A series of 4 cases of FL that contained diffuse areas of the large cell component was included as well with the same objective.
Immunohistochemistry Techniques
Immunohistochemistry detection of p16/INK4A was performed by incubating tissue sections overnight at 4°C with p16/INK4A monoclonal antibody (Calbiochem, Oncogene Research Products) diluted at 1:3000. After incubation with the primary antibody, immunodetection was performed with biotinylated anti-mouse immunoglobulins, followed by peroxidase-labeled streptavidin (Histostain Plus; Zymed, San Francisco, CA) with diaminobenzidine chromogen as substrate. Quick washes with Chemmate washing buffers (Dakopatts) between successive incubations were included to obtain a clean background.
For double-labeling experiments, after p16 immunodetection, an incubation with antibodies for CD20 (L26, Dakopatts) and CD3 (Polyclonal, Dakopatts) was performed, followed by incubation with the EnVision (Dakopatts) system, alkaline phosphatase, and Fast Red chromogen as substrate.
Incubation omitting the specific antibody, as well as with unrelated antibodies, was used as a control of the technique.
Results were interpreted according to established criteria.24,25 Briefly, p16/INK4A protein expression was considered normal when p16/INK4A nuclear immunostaining was present in all areas. The absence of nuclear staining in part or all of a tissue section was only considered to be a loss of p16/INK4A protein expression when the interspersed nuclei of reactive cells (lymphocytes, endothelial cells) displaying p16/INK4A expression were observed and used as an internal positive control. A partial loss of p16/INK4A staining was considered to have occurred when areas of preserved staining alternated with others showing regional loss of staining by the tumor cells.
Double Immunolabeling and Laser Scanning Confocal Microscopy
Tissue sections were also stained using double labeling with both mouse monoclonal antibody anti-p16 DCS-50.1 (Calbiochem, Oncogene Research Products) diluted 1:3000 and rabbit polyclonal anti-CD3 diluted 1:50 (Dakopatts). After simultaneous overnight incubation at 4°C with primary antibodies, sections were incubated with biotin-conjugated donkey anti-rabbit immunoglobulin G (1:50, Jackson ImmunoResearch Laboratories, West Grove, PA) and goat anti-mouse immunoglobulin G (1:50, Jackson ImmunoResearch Laboratories). After washing in Tris-buffered saline, sections were incubated with streptavidin Cy2 (1:50, Amersham) and Cy3-conjugated donkey anti-goat immunoglobulin G (1:50, Jackson ImmunoResearch Laboratories). Sections were mounted with glycerol and examined with an MRC 1024 Bio-Rad confocal system (Bio-Rad, Richmond, CA) mounted on a Zeiss Axiovert 135 microscope (Zeiss, Oberkochen, Germany). A 25-mw multilinea argon laser producing two major lines at 488 and 514 nm, was used. Images were stored on magnetic optical disks. Series of images were processed with the Lasersharp software package (Bio-Rad).
Molecular Studies
A total of 26 lymphoma DNA samples were included in the study, including cases with normal and absent p16/INK4A protein expression. Cases were included at random on the basis of the availability of frozen material for DNA extraction. Each tissue fragment used for molecular study was paralleled by another equivalent fragment that was processed for routine histology. DNA from reactive tonsils and PBLs from healthy donors were also included in the study.
Polymerase Chain Reaction-Single-Strand Conformational Polymorphism
Single-strand conformational polymorphism (SSCP) analysis was used to screen for p16/INK4A gene mutations, according to a slightly modified protocol of the type previously reported by Orita et al.26 We analyzed exons 1 and 2 of the CDKN2A gene, comprising 97% of the coding sequence. One hundred ng was amplified with 30 pmol of each primer, 200 µmol/L deoxynucleotide triphosphates, 5% dimethylsulfoxide, 1 µCi of 32P (Amersham), and 0.5 U of Taq polymerase with the primers previously described by Kamb et al.6 Twenty-five cycles of the polymerase chain reaction (PCR) at 94°C, 60°C, and 72°C for 20 seconds, 30 seconds, and 1 minute, respectively, with five previous cycles at a 68°C annealing temperature were performed in a Thermal cycler (Perkin-Elmer Corp., Norwalk, CT). The products were diluted in formamide buffer and electrophoresed at room temperature and at 10°C, with and without 10% of glycerol, for 16 to 18 hours at 7 to 12 W.
Direct Sequencing
Sequences of samples showing mobility shifts by PCR-SSCP analysis were confirmed by direct sequencing with an Automated DNA Sequencer ABI PRISM 310 Genetic Analyzer (Perkin Elmer) according to the manufacturer's instructions.
Methylation
PCR was used to determine the methylation status of the first exon of p16/INK4A, as described by Martinez-Delgado et al.27 Briefly, 100 ng of DNA digested by EagI enzyme was amplified in a multiple PCR for exons 1 and 2 of the p16/INK4A gene. Products were resolved on a 2% Nusieve agarose gel, blotted onto nylon membranes, and hybridized to digoxigenin-labeled internal oligonucleotide for exons 1 and 2. Oligonucleotides sequences were as follows: 5'-CCAGGCATCGCGCACGTCCAG-3' for exon 1 and 5'-AGGAGGTGCGGGCGCTGCTG-3' for exon 2. After digestion, only methylated tumors showed amplification of exon 1. Southern blot analysis was used to confirm the methylation status of the p16/INK4A, using restriction enzymes EcoRI and SacII in a double digestion. SacII does not cut DNA when there is methylation of the restriction site. Five µg of DNA was digested using 40 units of EcoRI and 60 units of SacII enzyme electrophoresed in 0.8% agarose gel and hybridized with a PCR-generated probe corresponding to exon 1 of p16/INK4A.
Analysis of Allelic Loss on Chromosome 9p21
DNA from lymphoid tumors and reactive lymphoid tissue was analyzed
for LOH or homozygous deletion by amplification of dinucleotide
repeat-containing sequences using PCR.16
Primers D9S171,
D9S1747, D9S1748, D9S1749, and interferon 
(IFNa) were obtained from
Research Genetics (Huntsville, AL). The amplification conditions were:
94°C for 30 seconds, 57°C for 40 seconds, and 72°C for 45
seconds, for 25 cycles. Radiolabeled, amplified products were separated
by electrophoresis in denaturing 8 mol/L urea-6% acrylamide gels,
followed by autoradiography. Homozygous deletion was scored when one or
more closely spaced markers demonstrated apparent retention of
heterozygosity when flanked by markers demonstrating clear
LOH.16
The signal intensity of fragments was determined by
densitometry (one-dimensional analysis and hand scanner settings;
Biomed Instruments, Zeineh Programs Fullerton, CA), scoring LOH
if an entire band was missing or the intensity of the band was reduced
below >40% of the normal band.27,28
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBLs had an undetectable p16/INK4A protein signal when studied by Western blot. After PHA stimulation, a 16-kd band corresponding to p16/INK4A protein was detected at 24 hours, with the signal attaining a moderate intensity at 72 hours.
The FCM expression of p16/INK4A in PHA-stimulated PBLs is illustrated
in Figure 1A
. The expression of p16/INK4A
on unstimulated PBLs was restricted to 11.26% of cells. However, after
24 hours of PHA stimulation, PBLs displayed a marked increase in
p16/INK4A reactivity, amounting to 39.53% of positive cells at 96
hours after PHA stimulation.
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. This increase was also found when
only the S and G2/M phases were analyzed. In Vivo Studies
Reactive Lymphoid Tissue
A diffuse mosaic pattern of p16/INK4A nuclear immunoreactivity was
observed in all of the different lymphoid compartments (germinal
centers, mantle cells, and interfollicular space), in different
proportions of cells. A proportion of roughly one-third of positive
cells was found in the different compartments, with a slight increase
in the germinal centers (Figure 2)
.
Similar results were obtained in the eight different samples analyzed.
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All 41 cases of NHL with a low growth fraction (chronic
lymphocytic leukemia, mantle cell lymphoma, FL, MALT low grade, and
splenic marginal zone lymphoma) showed preserved normal p16/INK4A
protein expression, characterized by a diffuse mosaic pattern of
p16/INK4A-positive nuclear immunoreactivity of the neoplastic cells. Of
the 71 NHLs with a high-growth fraction, p16/INK4A protein expression
was maintained in 30 cases (42%) and had undergone alteration in the
remaining 41 cases (58%) because of partial (14 cases, 20%) or total
loss (27 cases, 38%) (Table 1
and Figure 2
).
|
. To confirm that positive
cells were tumor cells and not bystander T cells, a
double-immunostaining procedure was performed that showed that most
p16-positive cells were B cells, whereas it was possible to find only
occasional p16-positive T cells (Figure 2)
. These results were
confirmed using double immunostaining by laser scanning confocal
microscopy with both CD3 and p16 antibodies, in both an FL and a
reactive germinal center, which detected p16 expression in both cell
types, although most p16-positive cells in the FL case were B cells
(Figure 3)
.
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. Macrophage
staining was usually weak or null, thus not precluding the recognition
of p16/INK4A expression in aggressive lymphomas.
For the analysis of an eventual p16/INK4A loss in tumor progression, a
large group of cases with both low- and high-grade components were
included (Table 2)
. All 37 MALT + DLBCL
cases showing transformation from the small cell/low growth component
into an aggressive variant displayed normal diffuse p16/INK4A protein
expression by the small cell areas. However, p16/INK4A expression by
the large cell/high growth component was normal in only 6 cases (16%)
and was found to be altered in the remaining 31 cases (84%), either by
focal (n = 8, 22%) or total loss
(n = 23, 62%) (Figure 2)
. Likewise, the 4 FL
cases showing areas of transformation into DBLCL presented normal
p16/INK4A expression by the low-grade component, whereas in the same
specimens, a p16/INK4A loss in the large cell component was found in
all 4 cases.
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Analysis of p16/INK4A Gene Mutation
Only one case was found to be mutated when 26 samples were
screened for p16/INK4A mutations (4%) (Table 3
and Figure 4
). Nucleotide sequences of the
CDKN2A gene with mobility shifts shown by PCR-SSCP analysis
were determined by direct sequencing. Four nucleotide changes were
identified in three different cases in exon 2. Three of the changes
correspond to a G
A transition in codon 148 that substitutes
ACGThr for GCGAla. This missense mutation has
previously been reported as a polymorphism.6
Cases L170 and
L169 show heterozygous polymorphism, whereas case BK20 shows hemi- or
homozygous polymorphism. The fourth change, found in a Burkitt lymphoma
(BK20), is a CG transversion at codon 129 (Figure 4)
. This
transversion produces a nonsense mutation TACTyr
TAGStop at the fourth ankyrin repeat domain of
CDKN2A. The wild-type allele can be observed (Figure 4B)
.
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The methylation status of two CpG islands in exon 1 of
p16/INK4A was analyzed by PCR and Southern blot using two
methylation-sensitive enzymes, EagI and SacII. We
examined a group of 23 cases with low and high proliferation indexes.
An abnormal pattern of methylation was detected in 6 of the 23 cases
studied (26%), all of which showed a high proliferation index. As is
shown in Figure 5
, exon 1 of
p16/INK4A was amplified only in cases methylated after
digestion with EagI. Nevertheless, exon 2, which lacks an
EagI site, was amplified in all cases. These experiments
were performed three times, and the same results were obtained in all
of them. For all of the cases except one (LL173), hypermethylation
status was confirmed with another methylation-sensitive enzyme
(SacII) detected by Southern blot. The diagnoses
corresponding to the methylated cases were MALT + DLBCL (three cases),
nodal DLBCL (two cases), and Burkitt's lymphoma (one case) (Table 3)
.
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Sixteen cases of NHL for which DNA from noninfiltrate tissue was
available were studied using markers for the microsatellites
surrounding the CDKN2A locus on 9p21. Allelic loss was found
in 3 of 16 cases (19%), all of them high-grade tumors (Table 3
and
Figure 6
). The genetic change detected by
microsatellite analysis was LOH in the three cases, with no cases
showing homozygous deletion. In the case MT51, it is not
possible to assume that the LOH detected at IFNa locus
implicates the p16 gene.
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No genetic alteration was found in the group of 13 cases with
preserved p16/INK4A expression (Table 4)
. Consequently, no genetic
alteration was identified in low-grade lymphomas. In contrast with this
finding, 9 of 13 cases with abnormal protein expression, with either
partial or total loss, showed different genetic changes (Tables 4 and 5)
. The most frequent alteration
identified in the cases with p16/INK4A loss of expression was CpG
island methylation (6 of 12 cases), followed by allelic loss (3 of 9
cases) and, more rarely, nonsense mutation (1 of 13 cases).
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Analysis of the cases did not show any specific relationship between individual genetic changes and histological diagnosis.
The partial expression of p16 protein in case BK20, with the detection
of apparent hemizygous polymorphism and heterozygous mutation (Figure 4B)
, suggests that this case lost one allele in the tumor and later in
evolution acquired the mutation in the left allele in only a part of
the tumor.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The expression of p16/INK4A was weak in PBLs as detected by FCM. However, after PHA stimulation, these cells display a rise in p16/INK4A expression, which is more accentuated in the S and G2-M phases of the cell cycle. This is consistent with previous studies showing that the level of p16/INK4A protein varies along the cell cycle.29 In concordance with these data, the samples from reactive lymphoid tissue (tonsils and lymph nodes) showed a variable intensity of staining in the different cellular microenvironments present in lymphoid tissue. Thus, although cells in all compartments express p16/INK4A, higher levels are found in those compartments where cell proliferation is increased (the germinal center and interfollicular area), in comparison with the weaker levels found in the mantle zone, where mainly resting lymphocytes are found.
Here we analyzed p16/INK4A protein expression in a series of 153 NHLs: 41 low-grade and 71 high-grade tumors, also including 41 cases with aggressive transformation from low to high grade. The pattern found in NHLs differs from that identified in reactive lymphoid tissue. Thus, although the expression of p16/INK4A is a constant feature in all 41 cases of low-grade NHL, it seems to be lacking in a significant proportion (41 of 71) of high-grade tumors due to total (27 cases) or partial loss (14 cases).
Our results show that this loss of p16/INK4A, besides being more frequent in aggressive cases, is strongly associated with tumor progression from low-grade to high-grade neoplasms. Thus, when the group of 41 cases showing areas of aggressive transformation from low- to high-grade component was analyzed, the low-grade component displayed normal p16/INK4A protein expression, which was confirmed by using double immunostaining for CD20 or CD3 and p16/INK4A, whereas the expression of p16/INK4A protein appears to be altered in the high-grade component in a majority of cases (35 of 41) either by partial (8 cases) or total (27 cases) loss. These data are roughly in agreement with those obtained for other types of tumors, in which the loss of p16/INK4A expression has been found to be associated with tumor progression.30-32 Indeed, the largest group in which p16/INK4A loss was found to be associated with tumor progression, large B-cell lymphoma presenting in mucosa, is claimed to frequently arise superimposed on low-grade MALT lymphomas, as an aggressive transformation of the same.34
Several previous studies have addressed the frequency of p16/INK4A molecular alterations in lymphoid malignancies. Frequent loss of p16/INK4A in T-acute lymphoblastic leukemia has been confirmed by different authors.21,34,35 Although the incidence of p16/INK4A homozygous deletion in NHL found to date ranged from 0 to 12%,15,17,18,35-37 it was significantly more frequent in high-grade neoplasms18,37 and lymphoid cell lines.8
The results shown here take advantage of the high sensitivity of immunohistochemical techniques for the detection of p16/INK4A loss, increasing the frequency of the finding of p16/INK4A loss in high-grade neoplasms while confirming its specific distribution in the subset of cases characterized by higher aggressiveness and showing that p16/INK4A loss is characteristically associated with tumor progression in NHLs.
Study of the genetic basis for this p16/INK4A loss disclosed some noticeable findings, mainly the existence of a relatively good correlation between immunohistochemistry and genetic studies and the high frequency of 5'-CpG island methylation. These observations are consistent with previous studies on the same subject. Thus, p16/INK4A mutation in NHLs has only rarely been detected,17 and the most frequent molecular alteration identified to date is homozygous deletion,17,21,34-38 although recent studies provide data that suggest that 5'-CpG methylation of p16/INK4A could play a significant role in p16/INK4A inactivation in lymphomas28,39 and myelomas,40 as has also been seen in other tumors.19,30
Thus, p16/INK4A inactivation seems to play a major role in tumor progression in lymphomas, in parallel with p53 mutations.37 Further efforts are, however, necessary to identify additional genetic changes in cell cycle-negative regulators that complement these findings and allow a more comprehensive explanation for NHL pathogenesis.
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Acknowledgements |
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Footnotes |
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RV and MS-B contributed equally to this work.
Supported by grants 94/0268 and 95/1805 from the Fondo de Investigaciones Sanitarias, and by the Fundación Ramón Areces, Spain. BM-D is supported by the Fundación Conchita Rábago Grant.
Accepted for publication June 26, 1998.
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M. Sanchez-Beato, A. I. Saez, I. C. Navas, P. Algara, M. Sol Mateo, R. Villuendas, F. Camacho, A. Sanchez-Aguilera, E. Sanchez, and M. A. Piris Overall Survival in Aggressive B-Cell Lymphomas Is Dependent on the Accumulation of Alterations in p53, p16, and p27 Am. J. Pathol., July 1, 2001; 159(1): 205 - 213. [Abstract] [Full Text] [PDF]
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S. Li, D. T. Ross, M. E. Kadin, P. O. Brown, and M. A. Wasik Comparative Genome-Scale Analysis of Gene Expression Profiles in T Cell Lymphoma Cells during Malignant Progression Using a Complementary DNA Microarray Am. J. Pathol., April 1, 2001; 158(4): 1231 - 1237. [Abstract] [Full Text] [PDF]
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 J. Serrano, S. U. Goebel, P. L. Peghini, I. A. Lubensky, F. Gibril, and R. T. Jensen Alterations in the p16INK4a/CDKN2A Tumor Suppressor Gene in Gastrinomas J. Clin. Endocrinol. Metab., November 1, 2000; 85(11): 4146 - 4156. [Abstract] [Full Text]
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I. C. Navas, P. L. Ortiz-Romero, R. Villuendas, P. Martinez, C. Garcia, E. Gomez, J. L. Rodriguez, D. Garcia, F. Vanaclocha, L. Iglesias, et al. p16INK4a Gene Alterations Are Frequent in Lesions of Mycosis Fungoides Am. J. Pathol., May 1, 2000; 156(5): 1565 - 1572. [Abstract] [Full Text] [PDF]
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A. Martin, F. Baran-Marzak, S. El Mansouri, C. Legendre, V. Leblond, F. Charlotte, F. Davi, D. Canioni, and M. Raphael Expression of p16/INK4a in Posttransplantation Lymphoproliferative Disorders Am. J. Pathol., May 1, 2000; 156(5): 1573 - 1579. [Abstract] [Full Text] [PDF]
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S. Kitazawa, R. Kitazawa, and S. Maeda Transcriptional Regulation of Rat Cyclin D1 Gene by CpG Methylation Status in Promoter Region J. Biol. Chem., October 1, 1999; 274(40): 28787 - 28793. [Abstract] [Full Text] [PDF]
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S. Kawano, C. W. Miller, A. F. Gombart, C. R. Bartram, Y. Matsuo, H. Asou, A. Sakashita, J. Said, E. Tatsumi, and H. P. Koeffler Loss of p73 Gene Expression in Leukemias/Lymphomas Due to Hypermethylation Blood, August 1, 1999; 94(3): 1113 - 1120. [Abstract] [Full Text] [PDF]
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M. Sanchez-Beato, F. I. Camacho, J. C. Martinez-Montero, A. I. Saez, R. Villuendas, L. Sanchez-Verde, J. F. Garcia, and M. A. Piris Anomalous High p27/KIP1 Expression in a Subset of Aggressive B-Cell Lymphomas Is Associated With Cyclin D3 Overexpression. p27/KIP1---Cyclin D3 Colocalization in Tumor Cells Blood, July 15, 1999; 94(2): 765 - 772. [Abstract] [Full Text] [PDF]
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