Delmer A, Tang R, Senamaud-Beaufort C, Paterlini P, Brechot C, Zittoun R
Hematology Department, Hôtel Dieu, Paris, France.
Leukemia. 1995 Jul;9(7):1240-5.
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.
细胞周期蛋白依赖性激酶4（cdk4）抑制剂（p16INK4/MTS1/CDKN2）基因最近被确定为一种假定的肿瘤抑制基因，因为在包括白血病在内的众多人类肿瘤细胞系中观察到纯合缺失的频率很高。然而，从未经培养的肿瘤样本中获得的结果引发了对这些发现相关性的讨论。我们使用逆转录聚合酶链反应（RT-PCR）和Southern印迹分析，在RNA和基因组水平上研究了p16INK4A基因在各种类型白血病中的情况：急性髓细胞白血病（AML）（n = 23）；急性淋巴细胞白血病（ALL）（n = 22）和B细胞慢性淋巴细胞增殖性疾病（CLPD）（n = 33）。仅在1/20的AML样本和2/23的CLPD样本中未发现p16INK4A mRNA表达。相反，在5/17的ALL病例中未检测到p16INK4A mRNA，另外7例中PCR产物的强度几乎检测不到，这可能与某些病例中正常细胞的污染有关。通过Southern印迹分析，在17例ALL病例中的6例（35%）中发现了p16INK4A基因的纯合缺失，其中4/6通过RT-PCR检测为阴性或弱阳性。所研究的5例AML和20例CLL样本均未发现p16INK4A缺失。在其中两例缺乏mRNA表达的病例中，p16INK4A外显子2的序列分析未显示点突变。我们的数据进一步证明，在血液系统恶性肿瘤中，ALL最有可能与p16INK4A失活相关，主要是通过纯合基因缺失。由于除ALL外的大多数血液系统恶性肿瘤很少与p16INK4A和视网膜母细胞瘤（Rb）基因改变相关，因此探索cdk4和cdk6的表达以确定p16INK4A/Rb/cdk4/cdk6调节环的破坏是否可能在其发病机制中起作用似乎是值得的。
