Immunohistochemical detection of apoptotic mechanisms in human lung carcinoma

Introduction:

The protein p53 induces cell growth arrest (apoptosis) in response to endo- or exogenous stimuli. P53 levels in normal cells is highly determined by MDM2 protein (murine double minute-2) – mediated degradation of p53. Mutation of TP53 (gene encodes a protein p53) is common in human malignancies and alters the conformation of p53. The result is a more stable protein which accumulates in nuclei of tumor cells with losing of function. Mutant p53 is stabilized, and by immunohistochemistry (IHC) it is possible to detect this form very clearly. As a potential marker of p53 function is expression of MDM2 protein. MDM2 overexpression represents at least one mechanism by which p53 function can be abrogated during tumorigenesis.

Patients and Methods:

Lung carcinoma samples were obtained from patients who underwent radical resection (lobectomy or pulmonectomy and lymphadectomy). The pathological diagnosis was based on WHO criteria. In our study we investigated expression of p53 and MDM2 protein that might improve IHC as a marker for p53 status. Immunohistochemistry (IHC) is a major method for observation of both proteins. Proteins were IHC detected in 136 samples of primary lung carcinoma. For IHC staining the following primary antibodies were used: anti-p53 protein (DO 7) (BioGenex Laboratories Inc.) and anti-MDM2 protein (SMP14: sc-965) (Santa Cruz Biotechnology Inc.). Immunostaining results of p53 positive samples were compared to IHC expression of MDM2 positive and MDM2 negative samples.

Results:

Strong brown nuclear staining was visible in p53 and MDM2 positive cells. Only 9 samples (7 %) were simultaneously p53 and MDM2 positive. In 46 (34 %) cases elevation of p53 was combined with MDM2 negative expression. Other tumour samples were either negative in both proteins (71/52 %), or p53 negative and MDM2 positive in 10 (7 %) tumour samples.

Conclusions:

Absence of p53 staining in most studies indicates absence of p53 mutation, and on the contrary, the positive expression of p53 is a sign of p53 mutations with losing of function. In our study in 34 % of cases extensively high level of p53 without increased level of MDM2 was identified. These are tumours with inactivating mutations that stabilize p53. On the other hand, tumours with high level of stabilized wild-type p53 protein and simultaneously with increased MDM2 staining (7 %) represent group with functional p53.

Acknowledgments: The work was supported by Grand VEGA 1/0224/12.