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Clinical Cancer Research Vol. 7, 3282-3288, October 2001
© 2001 American Association for Cancer Research

Regular Articles

Functional Impairment of Melanoma-associated p16INK4a Mutants in Melanoma Cells despite Retention of Cyclin-dependent Kinase 4 Binding1

Therese M. Becker2, Helen Rizos, Richard F. Kefford and Graham J. Mann

Westmead Institute for Cancer Research, University of Sydney at Westmead Millennium Institute, Westmead Hospital, NSW 2145, Australia


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: Melanoma-associated germ-line mutations affecting the tumor suppressor and cyclin-dependent kinase (CDK) inhibitor, CDKN2A/p16INK4a have been identified in >100 melanoma-prone families. To predict the melanoma risk for carriers of specific mutations, it is useful to test the function of the mutant proteins in biochemical assays; however, it is unclear how well these results correlate with their cellular effects. We examined the relationship between loss of CDK binding by mutant proteins and various measures of cellular growth in melanoma cells.

Experimental Design: The cellular activities of four melanoma-associated p16INK4a mutations (Arg24Pro, Ala36Pro, Met53Ile, and Val126Asp) were compared by use of inducible expression in stably transfected melanoma cells, deficient in expression of the endogenous protein, and compared with their ability to bind CDK4.

Results: The cell cycle-inhibitory activity of all of the mutants was profoundly reduced, and partially retained capacity for CDK4 binding in functional assays did not correlate with significant preservation of cell cycle-regulatory function.

Conclusion: Testing of p16INK4a interactions with CDKs in protein-binding assays is an unreliable predictor of mutant p16INK4a function in cells. In addition to exhibiting reduced stability, these mutant proteins may also be defective in interaction with cellular targets other than CDKs.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The CDKN2A gene on chromosome band 9p21 encodes the CDK3 inhibitor p16INK4a. Somatic alterations affecting this gene occur in >50% of human tumors (1 , 2) . Inherited CDKN2A mutations are associated with susceptibility to melanoma and, in some cases, pancreatic carcinoma (3 , 4) . To date, at least 48 germ-line p16INK4a mutations have been identified in >100 melanoma-prone families world-wide. Although these mutations may segregate closely with the disease in melanoma-prone families, demonstration of functional defects in the p16INK4a protein remains clinically important in distinguishing disease-associated mutations from population polymorphisms. p16INK4a was initially identified as a binding partner of CDK4 (5) . Although several functions of p16INK4a have been described in recent years, including the repression of the transcription factor NF{kappa}B (6) , it is widely assumed that the main role of p16INK4a is to inhibit CDKs. In particular, p16INK4a binds to and inhibits the activity of CDK4 and CDK6. These kinases regulate the progression of eukaryotic cells through the G1 phase of the cell cycle. CDK4 and CDK6 initiate the phosphorylation of pRb, which permits cells to enter DNA replication in S phase. CDK4 may also be involved in cell cycle progression through the G2 cell cycle phase and into mitosis (M phase; Ref. 7 ).

To investigate various p16INK4a mutations for their ability to bind and inhibit CDK4 and CDK6, a number of CDK-binding and kinase assays have been applied. These assays used p16INK4a and CDKs fused to GST (8) , polyhistidines (9) , or proteins translated in vitro (9, 10, 11) . Alternatively, in the yeast two-hybrid system, the proteins were fused to regulatory yeast peptides (12, 13, 14) . These tests confirmed that most cancer-associated p16INK4a mutations are functionally impaired. However, different assays revealed variable degrees of functional loss for identical p16INK4a mutations. For example, the Gly101Trp mutation is strongly associated with melanoma in >20 families world-wide, but its cyclin D1/CDK4 inhibitory activity in different functional assays ranged from 5 to 73% of the wild-type protein (8 , 9 , 12 , 14) . Functional deficiency of p16INK4a mutant proteins has also been shown after the gene or protein was introduced into a number of nonmelanoma and melanoma cell lines (15 , 16) . However, these studies were also limited because the cell cycle-inhibitory effect of functional p16INK4a selects against cells expressing the transgene and thereby affects long-term studies. This growth-inhibitory effect can be overcome by use of an inducible expression system, and Stone et al. (17) reported a detailed study of the effects of induced wild-type p16INK4a expression in melanoma cells. Consequently, we chose to thoroughly assess the function of melanoma-associated mutant p16INK4a proteins by stably introducing inducible constructs of the wild-type or mutant CDKN2A genes into melanoma cells lacking endogenous p16INK4a. The functional loss of all tested melanoma-associated p16INK4a mutations was much greater than CDK4 binding studies suggested. We postulate that these mutants are defective in interaction with cellular targets additional to CDKs.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
CDKN2A cDNA and Mutagenesis.
The originally cloned CDKN2A cDNA (5) was extended to full length (18) by PCR. Overlapping PCR, modified from Aiyar and Leis (19) , was used to engineer four p16INK4a mutations. The Arg24Pro (G071C) and Ala36Pro (G106C) variants were originally identified in our laboratory (20 , 21) , and the Arg24Pro mutation has now been reported in nine melanoma-prone families world-wide (11 , 13 , 20, 21, 22, 23, 24, 25) . The Met53Ile (G159C) variant was also originally detected in an Australian melanoma kindred (26) , and melanoma association has now been reported in 12 families world-wide (11 , 13 , 21 , 22 , 24 , 26, 27, 28) . The Val126Asp (T377A) variant was one of the first CDKN2A mutations identified; its function has been examined with different CDK binding and kinase assays and after introduction into cell lines. It has been associated with melanoma in at least three families world-wide (24 , 29 , 30) .

Protein Expression in Bacteria.
CDKN2A cDNA was cloned into the pGEX-5X-I vector (Amersham Pharmacia), and the insert and adjacent vector sequences were completely sequenced. Apart from the inserted point mutations, all CDKN2A constructs had identical sequences. GST-fusion p16INK4a (GST-p16INK4a) was expressed in Escherichia coli (JM109), purified using glutathione-Sepharose beads (as described by the supplier) and stored in glycerol lysis buffer [25% glycerol, 100 mM NaCl, 1% Triton, 1 mM DTT, 1 mM EDTA, 50 mM Tris (pH 8)]. CDK4 cDNA was obtained by reverse transcription-PCR from total RNA derived from human lymphoblastoid cells. The CDK4 cDNA insert was cloned into the pQE31 vector (Qiagen) and completely sequenced. CDK4 fused to six histidine residues (His-CDK4) was expressed in E. coli (BL21).

p16INK4a CDK4 Binding Assays.
Equal amounts of wild-type GST-p16INK4a, mutant GST-p16INK4a, or GST alone were immobilized on glutathione-Sepharose beads, and excess proteins were washed away. Bacterial lysate, containing excess levels of expressed His-CDK4, was added to the protein-coated Sepharose beads. The final binding buffer concentrations were as follows: 50 mM Tris (pH 8), 8.3% glycerol, 0.33% Triton, 33.3 mM NaCl, 0.33 mM DTT, and 0.33 mM EDTA. Incubation was performed for 1.5 h at room temperature or at 37°C with gentle agitation. The Sepharose beads were then washed three times with binding buffer. SDS-PAGE loading buffer was added, and samples were boiled for 5 min. Proteins were separated by 12.5% SDS-PAGE and transferred to a nitrocellulose membrane. CDK4 was detected by Western analysis with the antibody, clone DCS-35 (Neomarkers).

Cell Culture and Transfection.
The human WMM1175 melanoma cell line was originally isolated from a s.c. metastatic tumor of an individual with a family history of melanoma. The WMM1175 cell line is homozygously deleted for the CDKN2A region on chromosome 9p21 (31) , but expresses wild-type CDK4 and pRb (32) . Cells were grown in DMEM (Trace) with 10% FCS (Trace). An IPTG-inducible mammalian expression system (Lac-switch system; Stratagene) was used to obtain melanoma cell clones carrying the stably integrated CDKN2A gene under IPTG-inducible expression control. A WMM1175 clone transfected with the lac repressor vector, p3'SS, and expressing high levels of the lac repressor protein was selected for further transfection (33) . Full-length CDKN2A cDNA (wild type or mutant) was cloned into the expression plasmid, pOP-RSVICAT, and transfected using the calcium phosphate precipitation method. Transfected cells were selected with hygromycin (250 µg/ml) and geneticin (500 µg/ml; Life Technologies, Inc.). Expression of the transgene in selected clones was induced by 4 mM IPTG. The growth rate was monitored over 7 days, cell cycle distribution was studied after 24, 48, 72, and 96 h of p16INK4a induction (Becton Dickinson FACScan), and colony-forming ability was tested 14 days after induction.

SA-ß-Galactosidase Staining.
Clones induced to express wild-type p16INK4a protein with 4 mM IPTG were screened for expression of SA-ß-galactosidase, as described (34) . Senescent human fibroblasts were used as controls for SA-ß-galactosidase staining.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CDK4-binding Activity of Wild-Type and Melanoma-associated Mutant p16INK4a Proteins.
Wild-type and melanoma-associated mutant p16INK4a fused to GST were purified and tested for their ability to bind His-tagged CDK4. The amount of His-CDK4 bound to GST-p16INK4a proteins was determined by immunostaining. As shown in Fig. 1Citation , His-CDK4 copurified in this assay only when p16INK4a was immobilized to glutathione-Sepharose. Both the wild-type and mutant GST-tagged p16INK4a proteins bound comparable amounts of CDK4 when the proteins were allowed to interact at 20°C (Fig. 1)Citation . Because CDK4 binding to several p16INK4a variants, including the Val126Asp mutation, has been reported to be temperature sensitive (9 , 35) , the binding assay was repeated at 37°C. Increasing the incubation temperature to 37°C led to decreased CDK4 binding by two p16INK4a mutants [Met53Ile (35–45% of wild type) and Val126Asp (20–25% of wild type)], whereas CDK4 binding of the two remaining mutant proteins (Arg24Pro and Ala36Pro) was similar to that observed with wild-type p16INK4a (Fig. 1)Citation .



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  		Fig. 1. CDK4 binding to p16INK4a variants. After binding to p16INK4a, His-CDK4 was detected by Western analysis. CDK4 copurified only in the presence of p16INK4a. At the incubation temperature of 20°C, comparable amounts of CDK4 had bound to p16INK4a and copurified with glutathione-Sepharose (top row, Lanes 6–10). Increasing the incubation temperature to 37°C decreased the CDK4-binding ability of two mutant p16INK4a proteins, Met53Ile (to 35–45%) and Val126Asp (to 20–25%), compared with the wild type. The other two mutations behaved as wild type under these conditions (bottom row, Lanes 6–10). Lane 1, His-CDK4 bacterial lysate (3% of the amount used in the binding experiments); Lane 2, protein marker; Lane 3, binding experiment without any GST-p16INK4a; Lane 4, binding experiment without any His-CDK4; Lane 5, binding experiment using unfused GST protein; Lane 6, wild-type p16INK4a binding experiment; Lane 7, Arg24Pro p16INK4a binding experiment; Lane 8, Ala36Pro p16INK4a binding experiment; Lane 9, Met53Ile p16INK4a binding experiment; Lane 10, Val126Asp p16INK4a binding experiment.

 
p16INK4a Expression in WMM1175 Melanoma Cells.
To determine the cellular function of melanoma-associated p16INK4a mutants, wild-type and mutant CDKN2A genes were stably introduced separately into the p16INK4a-null WMM1175 melanoma cell line. The use of an inducible mammalian expression system for controlled p16INK4a expression allowed us to avoid growth arrest of the transfectants during the selection process. Selected CDKN2A-transfected WMM1175 clones expressed the protein on induction with IPTG. Titration experiments showed no detectable p16INK4a expression below 10 µM and maximal expression at 4 mM IPTG (data not shown).

Effects of Wild-Type p16INK4a Expression in Melanoma Cells.
The expression of wild-type p16INK4a produced major changes in the transfected WMM1175 cells. Cells were growth-arrested within 24 h of transgene induction, and this was accompanied by a strong decrease of cell numbers in S phase and a clear G1-phase block. The S-phase reduction and G1 block persisted through all subsequent time points (48, 72, and 96 h after p16INK4a induction) as long as IPTG remained present. After 48 h, a decrease in the number of G2-phase cells was also apparent as cells had traversed through G2 and accumulated in G1 phase. This shows that p16INK4a induces potent G1 arrest but does not affect the G2-M transition in WMM1175 under these conditions (Fig. 2)Citation .



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  		Fig. 2. Effects of p16INK4a expression on cell growth of WMM1175 melanoma cells. Growth curves show that wild-type p16INK4a expression stops cell growth after 24 h and up to 7 days postinduction (clone p16+++A4 shown). Expression of the mutations Arg24Pro (clone Arg24Pro+++A1 shown), Ala36Pro (clone A36Pro++A4 shown), and Met53Ile (clone Met53Ile++A8 shown) had only a marginal effect on the growth rate over 7 days, whereas expression of the Val126Asp (clone Val126Asp++A6 shown) mutation had no effect. Cell cycle distribution was determined after 48 h in 4 mM IPTG-treated (+) and mock-induced cells (-). Induction of the wild-type protein induced G1 growth arrest, which corresponded to a decrease of cell numbers in S phase and some reduction of cell numbers in G2 phase. Cell cycle distribution for other time points (24, 72, and 96 h) was very similar, with the G2 reduction not apparent before 48 h. Mutant p16INK4a expression caused only minor changes in cell cycle distribution.

 
Clonogenicity was abolished completely in WMM1175 cells with induced wild-type p16INK4a expression when compared with the parent cell line or selected p16INK4a-negative clones (Table 1)Citation . Withdrawal of IPTG from p16INK4a growth-arrested cells led to recovery, and cells reentered the cell cycle within 24–48 h (data not shown). p16INK4a-induced growth arrest was unchanged, and cells did not lose viability when IPTG induction was maintained in the medium for up to 3 weeks. The morphology of wild-type p16INK4a-inducible cells started to change after 24 h of p16INK4a expression: cells initially became more dendritic, gradually enlarged, and acquired a flattened granular phenotype, suggesting that these cells might have entered senescence. However, histochemical analysis for the presence of SA-ß-galactosidase, a marker for cellular senescence, was negative. In contrast, senescent human fibroblasts stained strongly positive in this assay (Fig. 3)Citation .


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  		Table 1 Effects of wild-type or mutant p16INK4a in WMM1175 melanoma cells and in CDK4 binding assays.

 


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  		Fig. 3. SA-ß-galactosidase staining of p16INK4a-induced WMM1175 melanoma cells. Cells induced for wild-type p16INK4a expression for 7 days stained negative for the senescence marker SA-ß-galactosidase despite a senescence-like phenotype. Senescent human fibroblasts used as a control for the SA-ß-galactosidase assay showed positive blue staining.

 
Effects of Mutant p16INK4a Expression in Melanoma Cells.
Expression screening of WMM1175 clones transfected with mutant CDKN2A cDNA for p16INK4a revealed fewer clones expressing p16INK4a protein; in addition, the levels of expression were usually lower than in clones transfected with wild-type p16INK4a. For additional experiments, clones expressing the highest mutant p16INK4a levels were selected; only the clone Arg24Pro+++A1 reached expression levels close to the levels of wild-type p16INK4a in p16+++ clones (Fig. 4)Citation .



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  		Fig. 4. Inducible p16INK4a expression in melanoma cell clones. Western analysis for p16INK4a before (-) and 10 h postinduction with 4 mM IPTG (+). Clones expressing various mutant p16INK4a constructs did not reach the same protein levels as the wild type. Lane 1, wild-type p16INK4a (clone p16+++A1); Lane 2, Ala36Pro (clone Ala36Pro++A4); Lane 3, Arg24Pro (clone Arg24Pro+++A1); Lane 4, Met53Ile (clone Met53Ile++A8); Lane 5, Val126Asp (clone Val126Asp++A6).

 
In contrast to the induced wild-type p16INK4a expression, none of the mutant proteins abolished clonogenicity and only the Met53Ile mutation led to a minor reduction (10%) in colony-forming ability. Furthermore, none of the mutant proteins induced the changes in cell morphology that were characteristic for wild-type p16INK4a expression. When more detailed measurements for cell proliferation were used, the four mutant p16INK4a proteins differed in their ability to induce cell cycle inhibition in the WMM1175 melanoma cell line. Induced expression of three p16INK4a mutations (Arg24Pro, Ala36Pro, and Met53Ile) had a reproducibly weak inhibitory effect on the growth rate over 7 days when compared with wild-type p16INK4a (Fig. 2Citation and summary in Table 1Citation ). These effects correlate closely with the consistently weaker G1 cell cycle block induced by the mutants when compared with the G1 arrest observed when the wild-type p16INK4a protein was induced (Fig. 2)Citation . The Val126Asp mutant showed more severe functional loss, failed to inhibit the growth of the WMM1175 cell line up to 7 days postinduction, and did not alter cell cycle distribution (Fig. 2Citation and Table 1Citation ).


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We initially tested the ability of wild-type and mutant p16INK4a-GST fusion proteins to bind His-CDK4. The data obtained for CDK4 binding correlated in part with the results reported by Parry and Peters (9) . Specifically, the CDK4-binding ability of the Val126Asp mutant decreased with increasing temperature. As expected from previous reports, the Met53Ile mutant, which is associated with melanoma in 12 families world-wide, had reduced capacity to bind CDK4 when compared with the wild-type protein (11 , 13) . In contrast, we found that the Arg24Pro mutant behaved normally in our CDK4 binding assay, despite cosegregation of this mutation with disease in nine melanoma-prone families. This contrasts with other studies that demonstrated reduced CDK-binding affinity for the Arg24Pro mutation (11 , 13) . It is possible that differences in the degree of functional loss are attributable to the type of fusion proteins used or the assay conditions applied, such as pH, temperature, or ionic strength. All of these parameters can influence protein conformation, stability, and surface charge and could thereby alter the degree of protein interactions. The Ala36Pro mutation, which has not been functionally tested previously, also showed wild-type CDK4 binding.

To compare the p16INK4a-CDK4 binding results with the effects of expressing wild-type and mutant p16INK4a variants in cells of melanocytic origin, we introduced the different CDKN2A constructs into p16-null melanoma cells. We applied an inducible mammalian expression system, which enabled us to obtain stable cell clones expressing either wild-type or mutant p16INK4a. In this model system, p16INK4a can interact directly or indirectly with a range of cellular targets such as CDK4, CDK6, CDK7, and NF{kappa}B. Furthermore, any decreased stability or expression of the mutant RNA or the protein will affect its function and can be directly assayed. This is an important advantage and overcomes the limitation of CDK-binding assays in which protein concentrations are usually equalized, possibly masking the effect of low physiological concentrations of a mutant gene product.

Expression of wild-type p16INK4a led to growth arrest within 24 h, which was shown to be attributable to G1-phase arrest. The growth arrest was maintained as long as IPTG was present and could be reversed by withdrawal of the inducer. These data confirm the findings by Stone et al. (17) . Additionally we showed that wild-type p16INK4a expression completely abolished colony-forming ability and that the senescence marker SA-ß-galactosidase is not detectable despite morphological changes characteristic of senescence. Although several reports have shown that p16INK4a is involved in inducing or maintaining the state of cellular senescence (36) , these data suggest that the up-regulation of SA-ß-galactosidase requires other pathways. These pathways may be disrupted in the WMM1175 melanoma cell line, and this possibility is under investigation at present.

Screening of the WMM1175 cell clones transfected with mutant CDKN2A revealed fewer clones accumulating the p16INK4a protein, and expression levels were generally lower. Because the constructs differed only by single point mutations, these results suggest that these melanoma-associated mutants show decreased RNA or protein stability. Ectopic expression of mutant p16INK4a protein in melanoma cells has previously been reported to be lower than wild type (37) . It is possible that this lower expression of mutant p16INK4a is an important determinant of function. There are potentially important implications of this for understanding possible effects of the heterozygous state on melanocytes of mutation carriers. For example, the decreased concentration of mutant protein in cells expressing both wild-type and mutant p16INK4a may help minimize any potential dominant-negative effect from the mutant protein as long the wild-type allele remains intact. Such a negative effect could occur if the mutant protein binds to wild-type protein and prevents its normal function, as has been described for some p53 mutants (38) . It would be important to determine whether such differences in protein concentrations are physiologically relevant in normal melanocytes.

In contrast to our CDK4 binding studies, the effects of mutant p16INK4a expression on melanoma cell growth were consistently different to those seen with the wild-type protein. None of the mutant p16INK4a proteins suppressed clonogenicity, apart from Met53Ile, which induced a small (10%) reduction. None of the striking changes in cell morphology observed with wild-type p16INK4a expression were seen in clones transfected with the mutant proteins (Table 1)Citation . In two previous studies, clonogenic assays were the main measurements for cell proliferation after p16INK4a expression. These studies had either introduced CDKN2A virally into fibroblasts with infection efficiencies of 50–80% (35) or had transfected plasmids constitutively expressing p16INK4a into melanoma cells, followed by a 14-day antibiotic selection period (despite the growth-inhibitory effect of p16INK4a; Ref. 37 ). The fact that, under those conditions, not all cells would have expressed the transgene may explain why the data in these studies did not show the dramatic difference between wild-type and mutant p16INK4a expression demonstrated here with stable p16INK4a-inducible cell clones. In contrast to our results, the authors of these studies (35 , 37) did not find complete inhibition of colony-forming ability by wild-type p16INK4a, and expression of mutant protein led to more variable results in the clonogenic assays.

Our use of inducible clones also permitted a more detailed examination of cell proliferation. Growth experiments over 7 days and examination of changes of cell cycle distribution at various time points after p16INK4a induction showed that three mutations (Arg24Pro, Ala36Pro, and Met53Ile) did retain small cell cycle-inhibitory function and that the Val126Asp mutation did not inhibit cell growth in any of the assays.

A comparison of our biochemical and cellular data revealed that the Ala36Pro and Arg24Pro mutations functioned as wild-type protein in the CDK4 binding assay but were functionally severely impaired in suppressing colony formation and induced only small growth reduction of WMM1175 cells. The Met53Ile mutation had weak CDK4 binding affinity and showed insignificant effects on the growth rate and cell cycle of WMM1175 cells, although it was the only mutation inhibiting colony formation to a small degree. CDK4 binding of the Val126Asp mutation was reduced to only 20–25%, and the functional loss in melanoma cells was even more severe because it showed no impact on colony-forming ability, growth rate, or cell cycle distribution. The Val126Asp mutation has been shown to have a disrupted secondary structure and the tendency to produce aggregates in vitro (39) . Walker et al. (37) observed a "speckled appearance" of this mutation after immunostaining, suggesting that aggregation of p16INK4A mutant proteins may be occurring intracellularly. This could help to explain the lack of activity of the Val126Asp protein in these melanoma cells.

Overall, these data indicate that testing of p16INK4a mutations with CDK binding and kinase assays is an unreliable and inconsistent guide to the functional loss of these proteins. These assays are incapable of reflecting the effect of the mutations on cellular expression levels and on cellular half-life; in addition, being based on binding to a limited number of known target molecules, such as CDK4 or CDK6, these assays fail to measure the combined effects of altered gene products on a variety of potential protein targets. The ability of p16INK4a mutations to differentially affect growth rate, cell cycle progression, and clonogenicity supports the concept that different mutations interfere with distinct functions of p16INK4a.

A number of alternative target proteins for p16INK4a have been identified in recent years. p16INK4a was shown to affect transcription by inhibition of phosphorylation of the CTD of TFIIH by CDK7 (40) . Recently this CDK7 inhibition was suggested to be linked to cell cycle regulation (41) . p16INK4a has also been shown to directly bind and inhibit the transcription factor NF{kappa}B (6) . NF{kappa}B regulates the expression of a number of genes essential for cell cycle control, such as p53 and cyclin D1 (42 , 43) . The p16INK4a interaction with specific target proteins may be influenced differently by various p16INK4a mutations. Indeed, it has been shown that NH2-terminal p16INK4a mutations, such as Arg24Pro, are impaired in their inhibition of TFIIH CTD phosphorylation by CDK7, whereas the Gly101Trp mutation located in the central region of p16INK4a has no effect on this particular function (41) . Interestingly, the Arg24Pro and Ala36Pro mutant proteins, which showed normal CDK4 binding in our assay but were functionally impaired in melanoma cells, carry mutations in the domain identified to be important for inhibition of TFIIH CTD phosphorylation by CDK7. Therefore, the examined p16INK4a mutations may be impaired for specific functions of p16INK4a, such as inhibition of the pRb pathway via CDK4 and/or the inhibition of TFIIH via CDK7. Furthermore, the mutation that reduced clonogenicity to a small degree, Met53Ile, may still inhibit TFIIH CTD phosphorylation by CDK7, which might specifically affect clonogenic survival. The presence of multiple targets for p16INK4a may explain why cellular based assays give more reliable data about a possible cancer risk associated with specific p16INK4a mutations than any protein binding test on its own.


   ACKNOWLEDGMENTS
 
We thank Axel Naumann (Children’s Medical Research Institute, Westmead, Australia) for the senescent fibroblasts, Shubhra Gupta (Westmead Institute for Cancer Research, Westmead, Australia) for the WMM1175 p3'SS clone, and Brian Gabrielli (Queensland Institute for Medical Research, Brisbane, Australia) for the p16INK4a antibody.


   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 Therese Becker was supported by scholarships of Sydney University (Overseas Postgraduate Research Scholarship, University Postgraduate Scholarship) and the Millennium Foundation, Westmead Hospital. The project was supported by the NSW Cancer Council, the National Health and Medical Research Council of Australia, and the Melanoma and Skin Cancer Research Institute, University of Sydney. Back

2 To whom requests for reprints should be addressed, Westmead Institute for Cancer Research, University of Sydney at Westmead Millennium Institute, Westmead Hospital, NSW 2145, Australia. Phone: 61 2 9845 9058; Fax: 61 2 9845 9102; E-mail: therese_becker{at}mail.wmi.usyd.edu.au Back

3 The abbreviations used are: CDK, cyclin-dependent kinase; NF{kappa}B, nuclear factor {kappa}B; pRb, retinoblastoma protein; GST, glutathione S-transferase; IPTG, isopropyl-1-thio-ß-D-galactopyranoside; SA, senescence-associated; CTD, COOH-terminal domain. Back

Received 4/16/01; revised 7/ 2/01; accepted 7/ 6/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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