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Alterations of cyclin-dependent kinase 4 inhibitor (p16INK4A/MTS1) gene structure and expression in acute
A Delmer1, R Tang, C Senamaud-Beaufort
1Hematology Department, Hôtel Dieu, Paris, France.
Abstract:
The cyclin-dependent kinase 4 (cdk4) inhibitor (p16INK4/MTS1/CDKN2) gene has been recently identified as a putative tumor suppressor gene because of the high frequency of homozygous deletion observed in numerous human tumor cell lines, including leukemias. However, results obtained from uncultured tumor samples have led to discussion of the relevance of these findings. Using reverse transcriptase polymerase chain reaction (RT-PCR) and Southern blot analysis, we have investigated p16INK4A gene at both RNA and genomic levels in various types of leukemias: acute myeloid leukemia (AML) (n = 23); acute lymphocytic leukemia (ALL) (n = 22) and B cell chronic lymphoproliferative disorders (CLPD) (n = 33). p16INK4A mRNA expression was not found in only 1/20 AML and 2/23 CLPD samples. Conversely, p16INK4A mRNA was not detected in 5/17 ALL cases, and intensity of PCR products were barely detectable in seven additional cases, possibly related to the contamination by normal cells in some cases. By Southern blotting, a homozygous deletion of p16INK4A gene was found in 6/17 ALL cases (35%) among which 4/6 were negative or weakly positive by RT-PCR assay. None of the five AML and 20 CLL samples studied had p16INK4A deletion. Sequence analysis of p16INK4A exon 2 did not show point mutation in two of these cases lacking mRNA expression. Our data provide further evidence that among hematological malignancies, ALL are the most likely to be associated with p16INK4A inactivation, mainly by homozygous gene deletion. Since most hematological malignancies-except ALL-are infrequently associated with p16INK4A and retinoblastoma (Rb) gene alteration it seems worthwhile to explore cdk4 and cdk6 expression to determine whether or not the disruption of the p16INK4A/Rb/cdk4/cdk6 regulatory loop might play a role in their pathogenesis.
Insights
The p16INK4A tumor suppressor gene is frequently inactivated by homozygous deletion in acute lymphocytic leukemia (ALL), but rarely in other leukemias. This suggests ALL is uniquely susceptible to p16INK4A alterations.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- The p16INK4A (CDKN2) gene is a known tumor suppressor.
- Homozygous deletion of p16INK4A is frequent in cancer cell lines, but its relevance in primary tumors is debated.
- p16INK4A regulates the cell cycle by inhibiting cyclin-dependent kinases 4 and 6 (cdk4/cdk6).
Purpose of the Study:
- To investigate p16INK4A gene alterations at RNA and genomic levels in various leukemias.
- To determine the frequency and mechanisms of p16INK4A inactivation in acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), and B cell chronic lymphoproliferative disorders (CLPD).
Main Methods:
- Reverse transcriptase polymerase chain reaction (RT-PCR) to assess p16INK4A mRNA expression.
- Southern blot analysis to detect homozygous deletion of the p16INK4A gene.
- Sequence analysis of p16INK4A exon 2.
Main Results:
- p16INK4A mRNA was detected in most AML and CLPD samples.
- p16INK4A mRNA was absent or weakly expressed in a significant proportion of ALL cases.
- Homozygous deletion of p16INK4A was identified in 35% of ALL cases, correlating with absent/weak mRNA expression.
- No p16INK4A deletions were found in AML or CLPD samples.
- No point mutations in p16INK4A exon 2 were found in cases lacking mRNA.
Conclusions:
- Acute lymphocytic leukemia (ALL) is the hematological malignancy most frequently associated with p16INK4A inactivation, primarily through homozygous gene deletion.
- AML and CLPD show infrequent p16INK4A alterations.
- Further investigation into cdk4 and cdk6 expression is warranted to understand the role of the p16INK4A/Rb/cdk4/cdk6 pathway in leukemogenesis.