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Clinical Cancer Research Vol. 6, 1219-1228, April 2000
© 2000 American Association for Cancer Research

Advances in Brief

Universal Inactivation of Both p16 and p15 but not Downstream Components Is an Essential Event in the Pathogenesis of T-Cell Acute Lymphoblastic Leukemia1

Motoko Omura-Minamisawa, Mitchell B. Diccianni, Ayse Batova, Ray C. Chang, Louis J. Bridgeman, John Yu, Jeanette Pullen, W. Paul Bowman and Alice L. Yu2

Department of Pediatrics/Hematology-Oncology, University of California, San Diego, California 92103 [M. O-M., M. B. D., A. B., R. C. C., L. J. B., A. L. Y.]; The Scripps Research Institute, San Diego, California 92037 [J. Y.]; The University of Mississippi, Jackson, Mississippi 39216 [J. P.]; and Cook Children’s Medical Center, Fort Worth, Texas 76104 [W. P. B.]


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
p16/p15 regulate the cell cycle pathway by inhibiting the cyclin Ds-CDK4/6 mediated phosphorylation of pRb. We reported previously that in T-cell acute lymphoblastic leukemia (T-ALL), p16 and p15 were frequently (~70%) inactivated at the DNA level by deletion, mutation, or hypermethylation. Therefore, we hypothesize that inactivation of the cell cycle regulatory pathway may be essential in the pathogenesis of T-ALL, and that the remaining T-ALL with a wild-type p16/p15 gene likely harbor inactivation of these genes at RNA or protein levels. Alternatively, the downstream components of the pathway including CDK4/6, cyclin Ds, and pRb may be deregulated. In 124 primary T-ALLs, we found inactivation of the p16 and p15 genes at the DNA level in 79 (64%) and 64 (52%) samples, respectively. Only 9 of the 45 samples with wild-type p16 expressed p16 protein, whereas the remaining 36 lacked p16 expression at the RNA or protein level. In the 60 samples with an intact p15 gene, only 2 expressed p15 mRNA, and the only one analyzed lacked p15 protein. Overall, the abrogation rates for p16 and p15 at DNA/RNA/protein levels were 93% (115 of 124) and 99% (123 of 124), respectively. Although no alterations were evident in cyclin Ds or CDK4/6, pRb was hyperphosphorylated in the majority of samples investigated. These findings strongly support that both p16 and p15 are specific targets in the deregulation of the cell cycle pathway in T-ALL and that the inactivation of these genes is most likely essential in the pathogenesis of this disease.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
The p16/p15-cyclin Ds/CDKs-pRb3 pathway plays a critical role in cell cycle progression. Protein complexes of D-type cyclins and CDK4/6 promote the G1-S phase transition (1 , 2) . This active complex induces the phosphorylation of pRb. The phosphorylated pRb releases transcriptional factors such as E2F, which activate the expression of genes essential for S-phase entry (3) . The ability of cyclin Ds-CDK4/6 protein complexes to phosphorylate pRb is prevented by CDK inhibitor proteins (4) . The CDK inhibitor family includes p16 (5 , 6) and p15 (7) , both of which reside on chromosome 9p21. Frequent alterations of the cell cycle regulatory pathway have been found in various human tumors, suggesting that deregulation of this pathway may be an essential event in their pathogenesis.

Deregulation of the cell cycle pathway can occur through two mechanisms: inactivation of CDK inhibitors themselves, or the deregulation of the downstream components. p16 inactivation by gene deletion (8 , 9) , mutation (10, 11, 12) , and hypermethylation of the promoter region (13 , 14) has been shown in a variety of tumors. In addition, a few studies have demonstrated inactivation of p16 at the transcriptional and posttranscriptional levels (12 , 15) , although the mechanisms remain unknown. p15 is also inactivated by deletion, mutation, or hypermethylation in various tumors (16, 17, 18) , although p15 expression in human cancers has not been examined in detail to date. As to the downstream components, various alterations have been reported. These include gene amplification/overexpression of cyclin D1 (19 , 20) , cyclin D2 (21) , cyclin E (22) , CDK4 (23 , 24) , and CDK6 (25) and mutations to CDK4/6 which inhibit p16 binding (26 , 27) . Deregulation of any of these components may result in pRb hyperphosphorylation and G1-S progression. In addition, alteration/deletion of the Rb gene may also yield a phenotype analogous to deregulation of the upstream components of the pathway.

Previous studies by us and others have shown that in approximately 50–70% of T-ALL, p16 is inactivated at the DNA level preferentially by homozygous gene deletion rather than by mutation (28, 29, 30, 31) . Furthermore, although the inactivation of p15 occurs at the same frequency as p16 in T-ALL, promoter hypermethylation contributes to a greater part of inactivation of p15 as compared with p16 (32 , 33) . The high rate of inactivation of these genes has led us to hypothesize that inactivation of p16/p15 may be an essential event in the pathogenesis of T-ALL and that the remaining 30% of T-ALL with an intact p16/p15 gene likely harbor either inactivation of p16/p15 at the RNA/protein levels or deregulation of the components downstream of p16/p15. To address this hypothesis, we investigated 124 leukemia samples obtained from T-ALL patients at the time of diagnosis for inactivation of p16 and p15 at DNA, RNA, and protein levels as well as deregulations of the downstream components of the pathway. Such information may offer a better understanding of the molecular biology of T-ALL and help to pinpoint the target molecule for the development of selective therapy.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Patient Population and Isolation of Primary T-ALL Cells.
Heparinized bone marrow or peripheral blood samples were obtained from 124 T-ALL patients who were enrolled in Pediatric Oncology Group ALL Biology protocols 9000 or 9400 at the time of diagnosis. Mononuclear cells were isolated from the samples by Ficoll-Hypaque density gradient centrifugation (Pharmacia Fine Chemicals, Piscataway, NJ). The content of lymphoblasts in the samples was generally >80%.

DNA Analysis.
Genomic DNA was isolated from T-ALL cells using the genomic DNA Isolation kit (Life Technologies, Inc., Gaithersburg, MD) or Trizol reagent (Life Technologies). The p15 and p16 genes were examined by Southern blot analysis, semiquantitative PCR, PCR-SSCP, and DNA sequence analysis, as described previously (28 , 34) . Analyses for promoter hypermethylation were also performed as described previously (32) .

RT-PCR.
Total RNA was isolated from 20–40 x 106 T-ALL cells using Trizol reagent (Life Technologies). RNA (2 µg) was reverse-transcribed into cDNA using the Superscript Preamplification System (Life Technologies), and 1–2 µl of cDNA were amplified under standard conditions as described previously (32 , 35) . Amplification of GAPDH was performed as a control. PCR products were resolved on a 2% agarose gel in 1x TAE (40 mM Tris-acetate, 1 mM EDTA).

Western Blot Analysis.
For Western blot analysis, we used 50–100 µg of protein in cell lysate prepared from 2.5–5.0 x 106 cells of fresh or cryopreserved samples. Western blot analysis of p16, pRb, ß-actin, and cyclin E was performed as described previously (35) . For the analysis of p15, CDK4, and CDK6 proteins, the blots were probed with anti-p15 (0.5 µg/ml; Neomarker, Fremont, CA), anti-CDK4 (0.125 µg/ml; Santa Cruz Biotechnology, Santa Cruz, CA), or anti-CDK6 antibody (0.5 µg/ml; Santa Cruz Biotechnology), respectively.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
To conduct a complete analysis of the p16/p15 cell cycle regulatory pathway, we must first identify T-ALL samples that retain at least one intact allele of p16 and/or p15 as the target samples for analysis of the gene expression. Those that expressed p16 and/or p15 were then further analyzed for the status of downstream components. Such systematic analyses were performed in 124 T-ALL diagnosis samples, which included 51 samples that had been examined previously and reported as to their p16/p15 gene and promoter hypermethylation status (28 , 32) . The remaining 73 samples were examined in the present study for p16/p15 gene alterations, and whenever a sufficient quantity of DNA was available, promoter hypermethylation was also studied. The combined results are summarized in Fig. 1, ACitation (p16) and B (p15).



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  		Fig. 1. Summary of inactivation of p16 (A) and p15 (B) in 124 T-ALL samples. p16 and p15 gene status and mRNA/protein expression were determined by PCR-SSCP, Southern blot analysis, RT-PCR, and Western blot analysis as described in "Materials and Methods."

 
Seventy-three of 124 (59%) T-ALL samples harbored homozygous deletion of the p16 gene. Five samples harbored DNA mutations in p16 exon 2, including four frameshift mutations and one mutation leading to a stop codon. An additional sample harbored gene rearrangement that resulted from a deletion encompassing the entire exon 2. Altogether, 79 (73 homozygous deletions, 5 mutations, and 1 rearrangement) of 124 (64%) T-ALL samples harbored alterations of both alleles of p16, whereas the remaining 45 retained at least one allele of the wild-type p16 gene (Fig. 1A).Citation Of those 45 samples, 18 [including 5 previously reported by Batova et al. (32) ] were analyzed for possible hypermethylation of the promoter region. Consistent with the published reports (32 , 33 , 36) , p16 promoter hypermethylation was infrequent with only a single case of hemizygous hypermethylation among the 18 samples investigated.

Analysis of the p15 gene revealed that 51 of 124 samples (41%) harbored homozygous deletions. Mutation analysis of p15 was not performed, with the exception of a few selected samples (see next section about p15 mRNA expression). Among the 73 samples retaining at least one allele of the p15 gene, 23 had been analyzed previously for promoter hypermethylation (32) . Thirteen of the 23 samples displayed p15 inactivation of both alleles through mechanisms related to promoter hyper-methylation (see footnote of Fig. 1B),Citation whereas the remaining 10 had at least one intact p15 allele (32) . Overall, 64 (51 homozygous deletions and 13 promoter hypermethylations) of 124 (52%) T-ALL samples harbored inactivation of both p15 alleles at the DNA level, whereas the remaining 60 retained at least one allele of the p15 gene (Fig. 1B).Citation

p16 and p15 mRNA Expression.
Upon the identification of 45 and 60 of 124 T-ALL samples having at least one allele of the p16 or p15 gene, respectively, the mRNA expressions of these genes were investigated by RT-PCR. For the purpose of comparison, five samples with p16 gene mutation and one with p16 gene rearrangement were also included in the analysis. The results are summarized in Fig. 1Citation , and representative samples are shown in Fig. 2Citation .



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  		Fig. 2. Expression of p16 and p15 mRNA in T-ALL samples. mRNA expression of p16 and p15 in T-ALL was determined by RT-PCR. All levels of gene expression are in comparison with that of GAPDH. Lane 1, normal T cells expressed p15 but not p16 mRNA; Lanes 2–6 (30570, 30106, 9338, 9316, and 7392) T-ALL samples with wild-type p16 and p15 genes; Lanes 7–8 (6972 and 8895), samples with mutation in p16 and intact p15 gene; Lane 9 (1701), sample with gene rearrangement of p16 and intact p15 gene; Lane 10 (9647), sample with homozygous deletion of both p16 and p15 genes shown as a negative control.

 
Normal mature T cells do not express p16 (Fig. 2Citation , Lane 1). In this study of the 45 samples with wild-type p16 gene, 16 expressed moderate to high levels of p16 mRNA (e.g., Lanes 2–4). The remaining 29 samples did not have detectable p16 mRNA (e.g., Lanes 5 and 6). In comparison, all five samples that harbored p16 mutation expressed high levels of p16 mRNA (e.g., Lanes 7 and 8). The one sample with a gene rearrangement expressed a truncated p16 transcript (Lane 9). These results are consistent with the findings in other cancers that transcription of p16 is often associated with alterations to the p16 gene itself or deregulation of pRb (12 , 37 , 38) .

In contrast to p16, p15 is expressed in normal mature T cells (Fig. 2Citation , Lane 1). However, among the 60 samples with at least one p15 allele, 58 did not have detectable levels of p15 mRNA expression (e.g., Lanes 2–7, and 9). The remaining two samples (nos. 8895 and 1393) expressed significant levels of p15 mRNA (e.g., no.8895 shown in Lane 8). Mutation analysis of exons 1 and 2 characterized p15 as wild type in these two samples (data not shown).

p16 and p15 Protein Expression.
p16 and p15 protein expression was examined by Western blot analysis in T-ALL samples that had a wild-type gene and expressed the corresponding mRNA. The results are summarized in Fig. 1Citation , and representative findings for p16 are shown in Fig. 3Citation . Only 9 of the 16 samples with wild-type p16 transcript expressed significant levels of p16 protein (e.g., nos. 30570 and 30106 shown in Figs. 2Citation and 3Citation , Lanes 2 and 3), whereas the remaining 7 samples displayed no or barely detectable levels of p16 protein (e.g., no. 9338 shown in Figs. 2Citation and 3Citation , Lane 4). As expected, none of samples that retained wild-type p16 gene but expressed no mRNA had detectable p16 protein (e.g., no. 9316 shown Figs. 2Citation and 3Citation , Lane 5). Neither did any of the four samples with frameshift mutations (e.g., no. 6972 shown in Figs. 2Citation and 3Citation , Lane 7). As for the two samples that expressed p15 mRNA, one (no. 1393) did not have a detectable level of p15 protein (data not shown), and the other (no.8895) was not analyzed because of an insufficient amount of protein. Of note, because the remaining one sample (no.8895) that was not analyzed for p15 protein harbored a nonsense mutation of p16, none of 124 T-ALL samples coexpressed both p16 and p15 protein.



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  		Fig. 3. Expression of p16 protein in T-ALL samples. Protein expression of p16 in T-ALL was determined by Western blot analysis. All levels of protein expression are in comparison with that of ß-actin. Lane 1, normal T cells did not express p16 protein; Lanes 2–4 (30570, 30106, and 9338), T-ALL samples that had wild-type p16 gene and expressed p16 mRNA; Lane 5 (9316), sample that had wild-type p16 gene and did not express p16 mRNA; Lane 6, PCL1691 (neuroblastoma cell line) shown as a positive control; Lane 7 (6972), sample that harbored a frameshift mutation and a high level of p16 mRNA expression.

 
Downstream Components of p16/p15 Cell Cycle Regulatory Pathway in T-ALL.
Although the majority of T-ALL samples did not express either p16 or p15, 9 samples expressed p16 protein. One additional sample (no. 8895) expressed p15 mRNA, although the protein expression status was unknown. Therefore, we considered the possibility of alterations of the components downstream of p16/p15, such as differential expression of cyclin Ds and CDK4/6 in these 10 samples. Although alterations of a single member of the cell cycle regulatory pathway seem to be sufficient to destabilize cell cycle regulation (37 , 39, 40, 41) , alterations of downstream components may occur in addition to p16/p15 inactivation (42 , 43) . Therefore, we also simultaneously analyzed 36 additional samples that lacked p16 and p15. For protein analyses of downstream components, the selection of samples from the 46 samples was based on the availability of sufficient quantity and quality of tumor cells.

Using RT-PCR, we examined these 46 for mRNA expression of the cyclin Ds; representative findings are shown in Fig. 4A.Citation Neither T-ALL samples nor normal T cells expressed cyclin D1 (data not shown). The majority of the samples expressed cyclin D3 (85%; 39 of 46), whereas cyclin D2 expression was detected in only 27 (59%) of the 46 samples. The expression levels of cyclin Ds were somewhat variable (Fig. 4A,Citation Lanes 2–7) but comparable with or lower than that of normal T cells (Lane 1). More importantly, no correlation between their expression patterns and p16/p15 status was evident (Fig. 4A).Citation



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  		Fig. 4. Expression of cyclin Ds (A) and CDK4/6 (B) in T-ALL samples. A, mRNA expression of cyclinD2/D3 in T-ALL was analyzed by RT-PCR. Lane 1, normal T cells; Lanes 2–7, T-ALL samples (6953, 437, 834, 6972, 7796, and 8246). GAPDH is shown as a control. 834 expressed p16 protein, whereas others harbored p16/p15 inactivation. B, CDK4 and CDK6 protein expression were determined by Western blot analysis. All levels of protein expression are in comparison with that of ß-actin. Lane 1, CDK4-amplified osteosarcoma cell line SJ SA-1; Lane 2, normal T cells; Lanes 3–6, T-ALL samples (6972, 8489, 854, and 1596). 1596 expressed p16 protein, whereas others harbored p16/p15 inactivation.

 
Western blot analysis for CDK4 and 6 was performed in 15 samples, including the 6 that expressed p16 protein and 9 that lack p16 and p15. Representative samples are shown in Fig. 4B.Citation The levels of CDK4/6 protein expression were variable (Fig. 4B,Citation Lanes 3–6). However, there was no evidence of gross elevation of their expression, as observed in an osteosarcoma cell line known to have gene amplification of CDK4 (Lane 1). Furthermore, there was no correlation between the expression levels of CDK4/6 and p16 status. We also considered the possibility of gene mutations to the p16 binding sites of CDK4 and CDK6 that could prevent p16 binding (26 , 27) . However, PCR/SSCP analysis revealed no mutations in these regions of CDK4/6 in any of the 46 samples examined, regardless of p16/p15 status (data not shown).

Cyclin E overexpression with the appearance of lower molecular isoforms has been reported in breast cancer (44) . The deregulation of cyclin E can bypass p16 cell cycle regulation and facilitate pRb phosphorylation (22) . However, no evidence of such cyclin E deregulation was found in a Western blot analysis of 19 samples, including the 4 that expressed p16 protein and 15 that lack p16 and p15 (data not shown).

Phosphorylation Status of pRb.
Lack of p16 and p15 expression is expected to result in the hyperphosphorylation of pRb, the end point of the G1-S cell cycle regulation. In T-ALL samples that retain p16 and p15 protein expression, pRb deregulation either by hyperphosphorylation or by loss of pRb may also yield a phenotype analogous to inactivation of p16/p15. In addition, expression of p16 has been shown to be tightly regulated by pRb status. To investigate the relationship between pRb and p16 status, Western blot analysis of pRb was performed in 42 of the 124 samples of which sufficient material was available. These 42 samples included 34 that lacked p16 at various levels and 8 of the 9 samples that expressed p16 protein. p15 was not expressed in any of the 42 samples. The results are illustrated in Fig. 5Citation and summarized in Table 1Citation .



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  		Fig. 5. Expression of pRb in T-ALL samples. Protein expression of p16 in T-ALL was determined by Western blot analysis. All levels of protein expression are in comparison with that of ß-actin. Lane 1, normal T cells expressed a low level of hypophosphorylated pRb; Lanes 3–5 and 7–12 (30570, 30612, 9647, 9338, 30106, 9041, 7392, 6972, and 669), T-ALL samples. Lanes 5, 7, and 10–12, samples lacked p16 at various levels; Lanes 3, 4, 8, and 9, samples expressed p16 protein. Lanes 2 and 6, IMR32 (neuroblastoma cell line) displayed only hyperphosphorylated pRb shown as a positive control. hyper-pRb, hyperphosphorylated Rb; hypo-pRb, hypophosphorylated Rb; wt, wild type; HD, homozygous deletion; mut, mutation.

 

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  		Table 1 Relationship between p16 and pRb status in T-ALL

 
Consistent with their resting state, normal mature T cells expressed only hypophosphorylated pRb (Fig. 5Citation , Lane1). Among the 34 samples that lacked p16 protein, 27 showed significant levels of pRb in the hyperphosphorylated state (Table 1Citation ; Fig. 5Citation , Lanes 5, 7, 10, and 11). In the remaining 7 samples, 2 did not have any detectable pRb (data not shown), and 5 showed predominantly hypophosphorylated pRb, although they expressed neither p16 nor p15 (e.g., Lane 12). Notably, of the 10 samples investigated for pRb status that retained the p16 wild-type gene but lacked p16 transcription, 9 expressed hyperphosphorylated pRb (e.g., Lane 10; see Table 1Citation , nos. 18–27). This is in direct contrast to normal resting T cells, where pRb is hypophosphorylated and p16 is not expressed. This is also contrary to the reported findings of a correlation between pRb hyperphosphorylation and p16 transcription (12 , 45 , 46) , suggesting that p16 transcription is inactivated in these T-ALL samples. Three of the eight samples that expressed p16 protein also displayed significant levels of the hyperphosphorylated form of pRb (e.g., Lanes, 3 and 9). The inability of p16 protein to inhibit pRb phosphorylation suggests that the p16 protein in these samples may not be operational. Of the remaining five samples, one did not express pRb (e.g., Lane 4). The other four samples expressed predominantly hypophosphorylated pRb (e.g., Lane 8), suggesting that p16 is functioning. Alternatively, it is possible that these four samples may harbor pRb mutation such as codon Cys-706->Phe, which is known to be sufficient to render pRb defective in phosphorylation and oncoprotein binding (47) . However, sequence analysis (48) of the four samples revealed that there was no mutation in two independent E1A/large T-binding regions (codon 393–572 and 646–772), which are common pRb mutation sites in other cancers (49) .


   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
In the present study of 124 primary T-ALL samples, we have found an overall abrogation rate of p16 and p15 at the DNA/RNA/protein levels in T-ALL to be as high as 93% (115 of 124; 79 DNA alterations, 29 with no mRNA expression, and 7 with no protein expression; Fig. 1A)Citation and 99% (123 of 124; 51 homozygous gene deletions, 13 promoter hypermethylations, 58 with no mRNA expression, and 1 with no protein expression; Fig. 1B),Citation respectively. Although alterations of the downstream components of p16/p15, including CDK4/6 and cyclin Ds, were not evident in T-ALL, pRb was hyperphosphorylated in the majority of samples investigated. These findings strongly support that both of p16 and p15 are specific targets in the deregulation of the cell cycle pathway in T-ALL and that the inactivation of these genes is most likely essential in the pathogenesis of this disease. Although p16 and p15 are inactivated in T-ALL through a number of different mechanisms including gene deletion, mutation, promoter hypermethylation, or transcriptional and translational inactivation, all appear to lead to the same biological consequence, deregulation of pRb and G1-S cell cycle transition.

p16 does not appear to regulate the cell cycle in normal mature T cells because p16 protein is undetectable (45 , 50) . A lack of p16 mRNA and protein expression in normal mature resting T cells was also confirmed in our present study (Figs. 2Citation and 3Citation , Lane 1). However, the expression status of p16 and p15 in proliferating T cells, which may be a more appropriate normal counterpart of T-ALL cells, is a different matter. Erickson et al. (51) have demonstrated recently the important role of p16/p15 for T-lymphocyte senescence. They showed that stimulated T cells accumulate p16 and p15 protein during population doublings and display high levels of these proteins as they enter into replicative senescence. Concomitant with p16/p15 accumulation, the senescent T cells showed increased binding of p16 to CDK6, decreased CDK kinase activity, and predominantly hypophosphorylated pRb. Other investigators have also reported a similar p16 accumulation in replicative senescence of fibroblasts (52 , 53) . Along the same line, the restoration of p16 expression in p16-deficient T-leukemic cell lines results in the inhibition of growth and the promotion of cell differentiation (54, 55, 56) . These findings suggest that p16 and p15 are key regulators in the senescence program in normal proliferating T cells, and inactivation of these genes facilitates malignant transformation of cells through alterations of the senescence program. They also support our conclusion that the inactivation of both p16 and p15 likely plays an essential role in the pathogenesis of T-ALL.

Previous studies have demonstrated that the p16 protein regulates pRb phosphorylation and that p16 transcription is stimulated by hyperphosphorylation or loss of pRb (12 , 45 , 46) . The inverse correlation between these two proteins may represent a tightly regulated feedback mechanism. In our study, such a correlation between pRb and p16 was observed in 13 T-ALL samples, which harbored pRb deregulation either by hyperphosphorylation or by loss of pRb (bold letters in Table 1Citation ; e.g., Fig. 5Citation , Lanes 3, 4, 7, 9, and 11). In these T-ALL samples, the feedback loops appear to be operating. On the other hand, among 29 samples that retain wild-type p16 gene but lack its transcription (Fig. 1A),Citation 9 of 10 investigated for pRb status showed pRb hyperphosphorylation (Table 1Citation , e.g., Fig. 5Citation , Lane 10). In comparison, normal mature resting cells did not exhibit p16 transcription and displayed only hypophosphorylated pRb (Figs. 2Citation and 5Citation , Lane 1). These findings distinguish the lack of p16 transcription in those T-ALL cells with a wild-type p16 gene from that in normal T-cells and may reflect an unknown mechanism that disrupts the induction of the p16 transcription through hyperphosphorylated pRb in T-ALL.

One possible mechanism for the loss of p16 transcription, despite a wild-type p16 gene, is promoter hypermethylation. However, we (32) and others (33 , 36) have demonstrated a very low frequency of p16 hypermethylation in T-ALL. Alternatively, we considered the possibility that mutations to the 5' upstream flanking region of the p16 gene may provide a new mechanism of p16 gene inactivation by interfering with the transcription. Therefore, sequence analysis of the 964-bp region upstream from the ATG start site of p16 was performed in 9 samples from these 29 samples that lack p16 transcription despite a wild-type p16 gene. This region has been shown to be sufficient for basal promoter activity (53) . However, sequence analysis failed to reveal alterations in this region in any of the samples examined (data not shown), suggesting that alterations of the 5' upstream region of p16 are also unlikely to be the mechanism of transcriptional loss of p16 in T-ALL.

In addition to p16 gene alterations and transcriptional inactivation of p16, we found seven T-ALL samples that had a wild-type p16 gene and mRNA expression but no protein (Fig. 1A).Citation Similar findings of p16 inactivation have been reported in lung and colon cancer cell lines (12) and in primary B-lineage leukemia cells (15) . It is possible that processing and/or translation of p16 mRNA may be disrupted. Alternatively, p16 protein may undergo rapid posttranslational degradation, as reported for p27 (57) . All together, only 9 of 124 (7%) T-ALL samples retained p16 protein. Among these 9 samples, three samples harbored significant levels of hyperphosphorylated pRb, suggesting that p16 protein is not functional, and one sample had no pRb expression. Taken together, 119 of 124 T-ALL samples (96%) harbored either abrogation of p16 or inactivation pRb, or both, strongly suggesting that alterations in the p16/p15-pRb cell cycle regulatory pathway may be an essential event in the pathogenesis of T-ALL.

Our findings suggest that both p16 and p15 are the essential targets for inactivation in T-ALL. Although p16 is predominately inactivated in many types of cancer, Herman et al. (16) showed that the p15 gene is selectively inactivated by hyper-methylation in acute myelogenous leukemia and glioma, suggesting an important tumor suppressor role for this gene in selected neoplasms. Furthermore, in normal tissues, expression of p15 is ubiquitous, whereas that of p16 is restricted to a few organs. p15, but not p16, is up-regulated by transforming growth factor ß, which is a potent cell growth inhibitor (7) . These data suggest that the two proteins likely play specific roles independent of each other, despite their identical functional behavior as CDK inhibitors (4) . We (32) and others (33 , 36) have demonstrated that in T-ALL, the p15 gene is inactivated not only through codeletion with p16 but also by mechanisms independent of p16, such as promoter hypermethylation. In the present study, we observed a lack of p15 mRNA expression, despite the presence of an unmethylated p15 gene in nine samples (Fig. 1B),Citation suggesting other mechanisms for inactivation of p15. Considering the possible unique roles of p16 and p15 and the high rate of coinactivation of these genes by independent mechanisms, we believe that both of them may be independent targets of the inactivation in T-ALL.

p16 and p14ARF share exon 2 in different leading frames, with alterations in this exon possibly affecting both genes (50 , 58 , 59) . Recent studies have demonstrated that p14ARF also regulates the cell cycle progression through interaction with p53 and MDM2 (60, 61, 62) . The high rate of p16 gene alterations in T-ALL suggests that inactivation of p14ARF may be as important or even more important than that of p16 in the pathogenesis of this disease. Our preliminary analysis revealed that the inactivation of p14ARF at the mRNA level is less frequent than that of p16, whereas inactivations of those genes at the DNA level occurred at approximately the same rates. Protein expression of p14ARF is now under investigation.

A similar high rate of p16 abrogation has been reported in primary pancreatic cancer, although the molecular level of inactivation differs from T-ALL; the p16 cell cycle regulatory pathway was abrogated in 100% of the 50 samples studied, all involving inactivation of p16 at the DNA level by mutation, deletion, or hypermethylation (14) . p15 status was not examined in this study. Inactivation of both p16 and p15 genes has been reported in 80 and 50%, respectively, of primary malignant lymphomas of the brain, although only 10 samples were examined and the samples were analyzed at the DNA level only (63) . In comparison, in other types of cancer such as primary sporadic melanomas, non-small cell lung cancers, and breast and colon cancers, the rates of p16 gene inactivation are much lower (~50%; Refs. 64 and 65 ), and those of p15 inactivation are usually less than those of p16 with few exceptions (16) . Overall, few investigations have included the systematic analysis of these genes at transcriptional and translational levels as performed in this study. Indeed, this is the first study to analyze both p16 and p15 thoroughly at DNA, RNA, and protein levels, as well as downstream components in primary T-ALL. This is also the first report to document that both p16 and p15 are abrogated nearly 100% in a large number of primary T-ALLs, which is the highest rate of inactivation of both genes in any neoplasm reported to date. This finding may reflect a unique molecular biological feature of T-ALL, and such information should provide an important key to develop molecularly targeted therapy in the future.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge Dr. Fred Behm for providing a portion of the T-ALL samples examined in this study from the Pediatric Oncology Group cell bank. We also thank Dr. Jean Wang for providing the pRb antibody and Dr. T. Nobori for providing p16 probe for Southern blot analysis.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by NIH Grants CA70391 (to A. L. Y.), LSA6124 (to A. L. Y.), CA79951 (to J. Y.), and LSA6226 (to J. Y.), the Cindy Matters Fund (to A. L. Y.), and in part by Grant MO1 RR00827 from the General Clinical Research Center program. Back

2 To whom requests for reprints should be addressed, at Department of Pediatrics/Hematology-Oncology, University of California San Diego Medical Center, 200 West Arbor Drive, San Diego, CA 92103-8447. Phone: (619) 543-8644; Fax: (619) 543-5413; E-mail: a1yu{at}ucsd.edu Back

3 The abbreviations used are: CDK, cyclin-dependent kinase; pRb, retinoblastoma protein; T-ALL, T-cell acute lymphoblastic leukemia; SSCP, single strand conformational polymorphism; RT-PCR, reverse transcription-PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Back

Received 10/27/99; revised 1/11/00; accepted 1/13/00.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

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