Apoptosis in cancer: therapeutic implications.

This review outlines the principal limitations of the mechanisms of active cell death (ACD, apoptosis) as the basis of tumorigenesis and the rationale of almost all therapies of malignancy. The concentration of cancer therapy in the direction of ACD induction is presented as both the result of progressive understanding of the mechanisms of apoptosis and that of the favourable tumor environment for ACD signal transmission. The latter property induces the by-stander killing of cancer cells, a fundamental mechanism because efficiency of all known methods of cancer treatment is far below 100%. Finally, recent results and hypotheses regarding cancer gene therapy based on final inductors of apoptosis and endogeneous ACD inhibitors in tumors are evaluated.

Graves-Basedow disease goiter: a model of Bax-Bcl2 regulated apoptosis.

This study demonstrates the involvement of a Bax-Bcl2-dependent apoptotic process in Graves-Basedow thyroid disease, a pathological condition known for its spontaneously oscillating evolution. A continuous series of 86 cases of surgically treated Graves' thyroid was evaluated for apoptotic cell content identified by histological criteria and confirmed by terminal desoxynucleotidyl transferase-mediated desoxyuridine triphosphate nick end-labeling (TUNEL). A significant correlation was found between tissue features of Graves' disease (epithelial hyperplasia, cellular hypertrophy, colloid content) and the amount of apoptotic cells. No correlation was found with lymphocytic infiltrates. Significantly, 11 cases (about 12% of the series) with high-level apoptosis displayed the typical features of active Graves' disease over all tissue sections. In contrast, cases with no detectable apoptosis exhibited regressive tissue features of Graves' disease. An intermediate group of cases was characterized by tissue heterogeneity with hyperactive foci, rich in apoptosis, alternating with regressive areas lacking apoptosis. In this group the participation of apoptosis to the remodeling of Graves' thyroid parenchyma, in a tight balance with cell proliferation, was best illustrated. Moreover, the thyroid follicle by accumulating apoptotic cells and bodies, allowed a tentative chronological ordering of apoptosis steps in correlation with Bax-Bcl2 tissue distribution and cellular pattern. Our observations suggest that the initiation of apoptosis corresponds to a loss of cellular cohesion, a drop in Bcl2 expression, and a delocalization of Bax from a putative Golgi storage location to a mitochondrial distribution.

Importance of DNA fragmentation in apoptosis with regard to TUNEL specificity.

In the absence of a universal specific molecular tracer of apoptosis, structural DNA alterations provide the basis of labeling systems: double-strand fragmentation for TUNEL (terminal transferase-mediated dUTP nick end-labeling), denaturation for poly (A) in situ hybridization, immunogenicity of single strand DNA, all methods which imply limited specificity due to the unavoidable presence of DNA breaks in virtually all cells. Thus, TUNEL application has been restrained to a narrow spectrum of sample conditions which has limited, in particular, retrospective surveys and apoptotic nuclei-protein double labelings. In the apoptotic nucleus two main obstacles intervene between TUNEL reagents and their targets: DNA hypercondensation and proteins around DNA. The former increases in the course of apoptosis and both are worsened by crosslinking and precipitating fixatives. This point out that TUNEL is an ambitious approach whose target, apoptotic DNA breaks, is less accessible than breaks occurring in non-apoptotic less compacted DNA. However, TUNEL has an advantage: the far greater degree of apoptotic DNA fragmentation. How to obtain a frank differential staining between apoptotic and non-apoptotic DNA? It appears that the answer relies on the pretreatment step and not in modifying the TUNEL staining protocol, which is optimal. Adapted pretreatments are able to circumvent accessibility obstacles and to extend TUNEL applicability to the most demanding conditions, those of archived tissue samples and of TUNEL--protein double labelings.

Antitumor activity of bax and p53 naked gene transfer in lung cancer: in vitro and in vivo analysis.

In vitro and in vivo data have demonstrated that virus-mediated p53 gene transfer can induce active cell death and lung tumor regression. In contrast, the therapeutic potential of bax, another apoptosis-inducing gene, has not been described. We compared p53 and bax cytotoxic effects by transient transfection of an average of 25 +/- 5% of the H-322 and H-358 bronchioloalveolar carcinoma cell lines in vitro. Under these conditions, bax expression killed 70 to 90% of the transfected cells whereas p53 killed only 40% of them. The killing activity of both genes involved apoptosis, as shown by TUNEL staining. Surprisingly, BrdU incorporation indicated that the cells that did resist Bax toxicity were blocked in the pre-S phase of the cell cycle, a result expected for p53 only. In vivo, repeated injections of naked DNA encoding Bax or p53 inhibited the growth of 4-mm preestablished H-322 tumors in nude mice. Growth retardation only, and not inhibition, was observed in H-358, a poorly transfectable and rapidly growing tumor. These results indicate that Bax and p53 share a similar, strong antitumor activity in vivo, even if the former is a more potent inducer of apoptosis in vitro.

Cell death and cancer: replacement of apoptotic genes and inactivation of death suppressor genes in therapy.

This review provides a critical evaluation of the increasing use of gene therapy in the treatment of malignancies to induce active cell death (ACD, apoptosis). This approach is consistent with the notion that cancer is an anomalous accumulation of cells largely resulting from diminished cell death. The review details the main genes potentially useful for therapy. Among these, p53 has received the majority of the investigators' attention and provided encouraging results. Even greater hope is offered by newly tried direct inducers of apoptosis, such as bax, bclXs and caspases. Another fruitful direction is the association of apoptosis-inducing gene transfer with radio- and chemotherapy, which are also inducers of ACD. There is a delicate balance between cell gain through mitosis and cell loss in neoplasia because spontaneous apoptosis is widely present in tumors. In fact, the tumor environment favors bystander cell killing which appears to be a fundamental mechanism insofar as the rate of observed cell mortality cannot be accounted for by the known methods of gene transduction with efficiencies far below 100%. We conclude that apoptosis offers a mainstream approach for cancer gene therapy since ACD is highly inducible and only limited gains in malignant cell apoptosis may displace tumors from growth to regression.

p53 mutant immunophenotype and deregulation of p53 transcription pathway (Bcl2, Bax, and Waf1) in precursor bronchial lesions of lung cancer.

Lung cancer is the end result of a multistep process in which genetic and molecular changes accompany, in an unknown temporal sequence, histological precursor (preinvasive) bronchial lesions. Biomarkers allowing prediction of the rate of progression of precursor lesions at different locations in the anatomical field may be clinically useful. Toward this aim, we analyzed, using immunohistochemistry, the expression of the p53 gene and of its transcriptional target genes bax, bcl2, and waf1 in preinvasive bronchial lesions from 69 patients with lung cancer and in similar lesions from 20 patients with no cancer progression. p53 accumulation occurred with increasing frequency, from 19% in mild dysplasia to 36% in moderate dysplasia and 59% in carcinoma in situ, and was exclusively observed in patients with p53-positive carcinoma. The higher frequency of the p53-positive immunophenotype in lesions adjacent to the p53-positive carcinoma, as compared to lesions distant from it, suggests that p53 mutant preneoplastic lesions had a higher rate of progression to invasion than did p53-negative lesions. Similar lesions in patients with no progression to lung cancer were all p53 negative. Bcl2 overexpression and Bax down-regulation, as shown by immunostaining, occurred in preinvasive lesions and were mainly maintained during invasion. The expressions of bax, bcl2, and waf1 did not correlate with p53 status. We conclude that p53 stabilization in preinvasive lesions has high predictive value for progression to invasion and that Bax/Bcl2 imbalance contributes to the clonal expansion during premalignant states.

TUNEL apoptotic cell detection in tissue sections: critical evaluation and improvement.

TUNEL, i.e., terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, has become a widely used staining method to assist in detection of apoptotic cells in tissue sections. However, despite its apparent simplicity, this technique has led to considerable disappointment because of its serious limitations in sensitivity and, even more, in specificity. We reviewed the limitations and artifacts of TUNEL and designed a comprehensive protocol to reassess the various procedures in use for five crosslinking and/or precipitating fixatives. By introducing microwave heating in extreme pH-value solutions (pH 3 for formalin and pH 10.6 for Bouin's fixative) coupled with proteolysis, we obtained an intense staining of 70-80% of apoptotic cells and bodies on archival tissue blocks, with little or no background. Owing to the enhanced sensitivity, early stages of apoptosis could be visualized and may enlarge our vision of the apoptotic cell beyond the mere image of shrinkage necrosis. We conclude that TUNEL remains a technique as useful as it is delicate, requiring critical interpretation of the staining. This study points out that, on archival tissues, despite the technical improvements we propose no protocol can be the final answer to all problems. Technique must be readjusted for any variation in tissue processing. However, step-by-step progress has rendered this method not only applicable but also performable within the constraints of archival surgical pathology specimens.

Clear cell tumor of the lung: an immunohistochemical and ultrastructural study supporting a pericytic differentiation.

Clear cell tumor ("sugar tumor") of the lung is a rare benign lesion with unclear histogenesis. It is composed of large cells with a clear cytoplasm rich in glycogen, blended with an abundant network of sinusoid-type vessels. We report two cases of sugar tumor, one of these lacking clearly demonstrable glycogen storage. In both, the tumor cells lacked keratin expression and were positive for vimentin and HMB 45, an antibody recognizing perivascular or myoid cell proliferation such as lymphangioleiomyomatosis and angiomyolipoma. The tumor cells were also immunoreactive for an endothelial cell marker, CD 34, but negative for Factor VIII or smooth muscle actin. Intercellular deposition of basal-like material was immunostained with Type IV collagen. At ultrastructural examination of one of these cases, tumor cells showing features of pericytes or poorly differentiated perivascular leiomyocytes encased in basement material were observed in close association with endothelial cells; their cytoplasm contained numerous membrane-bound glycogen and pinocytic vesicles. We conclude that on the basis of immunohistochemical and ultrastructural phenotype, sugar tumor presents pericytic features and that glycogen storage is not a constant feature of these benign tumors.

Multifocal alveolar hyperplasia associated with lymphangioleiomyomatosis in tuberous sclerosis.

Multifocal alveolar hyperplasia associated with pulmonary lymphangioleiomyomatosis is reported in a 21-year-old woman with tuberous sclerosis. Beside the cystic lesions of lymphangioleiomyomatosis, the tomography showed nodules up to 8 mm in both upper lobes. A proliferation of type II pneumonocytes and Clara cells lining the alveolar walls in an adenoma-like pattern was observed. Nuclear atypia, mitoses and necrosis were not observed, providing evidence against multicentric bronchioloalveolar carcinoma or micronodular atypical alveolar adenomatous hyperplasia. Whereas the lymphangioleiomyomatosis lesions showed strong positivity for HMB45 and expressed oestrogen and progesterone receptors, the alveolar hyperplasia was negative for these markers as it was for carcinoembryonic antigen, p53 and MIB1 antibodies. Multifocal alveolar hyperplasia in tuberous sclerosis is probably a benign hamartomatous lesion in our case without progression on a 2-year follow-up. Its histogenesis is unknown, but is possibly related to chromosome instability.

Apoptosis-related factors p53, Bcl2, and Bax in neuroendocrine lung tumors.

Neuroendocrine (NE) lung tumors comprise four classes of progressive aggressiveness for which proliferation and apoptosis rates could both contribute to their distinctive behavior. As p53 mutations may favor escape from apoptosis through changes in Bcl2-Bax expression balance, which are survival and apoptotic genes, respectively, we studied 121 NE lung tumors (16 typical carcinoids (TC), 5 atypical carcinoids (AC), 29 large-cell NE carcinomas (LCNECs), and 71 small-cell lung carcinomas (SCLCs) using immunohistochemistry. We quantified apoptosis by terminal-deoxynucleotidyl-transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) in 31 of these cases. There was a significant increase of p53 mutant immunophenotype (defined as immunoreactivity with at least two antibodies for at least 20% of tumor cells) between atypical/typical carcinoids group and the LCNEC/SCLC group (P = 0.0003). There was an inverse correlation (P < 0.0001) between the scores of Bax and Bcl2 expression in individual tumors and a significant inversion of the Bcl2. Bax ratio between low-grade (typical and atypical carcinoids) and high-grade (LCNECs and SCLCs) tumors with a predominant Bax expression in the first group and predominant Bcl2 expression in the second. Whereas carcinoids had variable apoptotic indexes, LCNECs had high indexes (1.3 to 6.8%), Bcl2 overexpression, Bax down-regulation, and Bcl2.Bax ratio > 1 correlated with lower apoptotic index in both LCNEC and the pool of LCNECs and SCLCs (P < 0.05) and a lower survival rate in the group of atypical and typical carcinoids and LCNECs (P < 0.002). The highest levels of Bcl2 expression and Bcl2.Bax ratios were associated with p53 mutant immunophenotype (P = 0.02). Our results suggest that aggressiveness in NE lung tumors could be linked, in addition to proliferation, to apoptosis-related factors.

In situ apoptotic cell labeling by the TUNEL method: improvement and evaluation on cell preparations.

TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) is a method of choice for rapid identification and quantification of the apoptotic cell fraction in cultured cell preparations. However, TUNEL application has been restricted to a narrow spectrum of sample conditions, and only detergents have been proposed as labeling enhancers. This study was aimed at extending TUNEL to variously fixed cells and improving TUNEL sensitivity by optimized pretreatments, the specificity being assessed by reference to the apoptotic morphology. Comparative TUNEL was performed with three protocols on CEM-C7 cells, a model of glucocorticoid-induced apoptosis. Samples were submitted to six modalities of fixation and TUNEL was performed after each of the following conditions: no pretreatment; detergent permeabilization; proteolytic digestion; microwave irradiation; and a recently published combination of the latter two. The proportion of TUNEL-stained elements within the cell fraction, with and without apoptotic morphology, was quantified. Our results showed that: (a) with an adequate pretreatment, reliable TUNEL can be obtained after each fixative tested; (b) detergent was inefficient in improving sensitivity; (c) whatever the fixation, microwave pretreatment provided the best TUNEL sensitivity without notable loss of specificity; (d) under adaptive technical conditions, TUNEL can be associated with detection of various proteins by double labeling; and (e) the existence of a limited population of intensely TUNEL-positive cells that lacked apoptotic morphology contributes to the current debate about a preapoptotic state.

Comparison of 35S and chemiluminescence for HPV in situ hybridization in carcinoma cell lines and on human cervical intraepithelial neoplasia.

For in situ hybridization (ISH), development of sensitive, nontoxic alternatives to the use of radioactivity is a constant concern. In this trend, and close to chromogenes and fluorophores, chemiluminescence appears an attractive method. A first positive experience in immunocytochemistry and in ISH, by using the enhanced luminol as luminogene substrate for horseradish peroxidase (HRP) led us to compare the sensitivity of 35S autoradiography and chemiluminescence. For this purpose, we used three human carcinoma cell lines, CaSki [400-600 copies of human papilloma virus (HPV) 16], HeLa (10-50 copies of HPV 18), and SiHa (1-5 copies of HPV 16), and 40 biopsy specimens of human cervical preneoplastic and neoplastic lesions. We performed ISH by using HPV cDNA biotin-labeled probes, detected by a two-step immunocytochemical reaction, the secondary antibodies being either 35S-labeled for autoradiography or HRP-labeled for chemiluminescence. An intensified CCD camera allowed acquisition of the luminescent signal. After only 10 min of photon accumulation, on cell line smears as well as on serial tissue sections, chemiluminescence gave comparable results to those obtained by a 3-week exposure for 35S autoradiography. A quantitative approach on cervical biopsy specimens confirmed this similar level of sensitivity by measuring the area of 35S- or chemiluminescence-stained nuclei. Our results indicate that chemiluminescence is a credible and perfectible alternative to radioisotopes for in situ detection of nucleic acids by hybridization.

Overexpression of nucleoside diphosphate/kinase A/nm23-H1 protein in human lung tumors: association with tumor progression in squamous carcinoma.

Levels of nm23-H1/nucleoside diphosphate/kinase A expression have been reported to correlate inversely with metastatic potential in some tumors but not in others. To clarify the role of nm23 in lung carcinoma, the genetic abnormalities of nucleoside diphosphate/kinase A/nm23-H1 were investigated at the DNA and protein levels. A series of 104 human lung tumors (42 neuroendocrine (NE) and 62 non-NE tumors) was analyzed for nm23-H1 protein expression by immunohistochemistry using one polyclonal and two monoclonal Ab and for genomic alterations using Southern blotting and single-strand conformation polymorphism. Overexpression of the nm23-H1 protein relative to the normal lung epithelia (pneumocyte and bronchial epithelial cells) was observed in 83% (35/42) of NE carcinomas and in 89% (55/62) of non-NE carcinomas. Eight of nine carcinoids exhibited an increased expression of nm23-H1 protein, suggesting that this overexpression of the nm23 protein is necessary for proliferation in any tumors. No significant correlation was found between nm23 staining and any clinicopathologic parameters in NE carcinoma or in adenocarcinoma. In squamous carcinoma, high levels of nm23-H1 protein expression were associated with tumor stage (p = 0.0036). Allelic deletion or genetic amplification was never found. No altered mobility was detected using single-strand conformation polymorphism analysis. These data show that nm23-H1 protein is overexpressed in a large number of lung tumors of all histologic types, in association with advanced tumor stage in squamous carcinoma. They also suggest that nm23-H1 might play a role in the progression of lung tumors rather than in antimetastatic function.

Alpha 2-macroglobulin: a binding protein for transforming growth factor-beta and various cytokines.

alpha 2-Macroglobulin (alpha 2M) is a large plasma glycoprotein that has long been known as an irreversible inhibitor of a variety of proteinases. More recently, it has been reported that numerous growth factors, cytokines and hormones bind to alpha 2M through diverse mechanisms. We review here a series of observations from our laboratory that support the concept that alpha 2M is a carrier protein for transforming growth factor-beta (TGF-beta) and allows this factor to act as an autocrine regulator of adrenocortical steroidogenic functions. alpha 2M was found to by synthesized and secreted by primary cultures of bovine adrenocortical cells in fairly large amounts (1 microgram/10(6) cells/24 h). TGF-beta is also secreted by this cell type, although under a latent form. Two distinct latent TGF-beta complexes have been characterized in adrenocortical cell conditioned medium, one of which is a complex between alpha 2M and TGF-beta. Although alpha 2M prevents the binding of TGF-beta to its membrane receptors, long-term incubation of alpha 2M with adrenocortical cells results in inhibition of cortisol production similar to that observed in the presence of TGF-beta alone. Taken together, these observations suggest that adrenocortical cells can release active TGF-beta from its latent complex with alpha 2M through an unknown mechanism. alpha 2M can therefore be considered as a TGF-beta carrier protein.

Comparison of seven bio- and chemiluminescent reagents for in situ detection of antigens and nucleic acids.

Bio- and chemiluminescence have proved sensitive enough to compete with chromogenic and radioisotopic tracers for in situ detection. However, they must also provide a discriminant morphological analysis of the specific signal. We have tested seven bio- or chemiluminescent reagents for tissue antigen and nucleic acid detection by immunocytochemistry (ICC) or in situ hybridization (ISH). They were based on luminescent detection of peroxidase, alkaline phosphatase, beta-galactosidase or xanthine oxidase. We also explored whether high molecular weight polymers could increase the spatial definition of the photon emission. An ICCD camera was used to collect the light signal provided by immunolabelling of endothelial cells and by ISH of human papilloma virus on cell smears. Among the enzyme-luminescent substrate combinations tested, the enhanced luminol chemiluminescence (ECL) gave the best resolution of the specific signal. The other systems were mainly hampered by a high diffusion of the reaction product over the tissue section. Unfortunately, in this case, the high molecular weight polymers tested were inefficient. However, the addition of polyvinylalcohol (PVA) or polyvinylpyrrolidone (PVP) significantly improved respectively the definition and intensity of ECL photon emission. We demonstrate that chemiluminescence gives a morphological resolution allowing histological examination. The extension of this new application, now depends on physicochemical adaptation of chemiluminescent reagents to the constraints of tissue detection.

Transforming growth factors beta stimulate both thrombospondin-1 and CISP/thrombospondin-2 synthesis by bovine adrenocortical cells.

We recently observed that adrenocortical cells secrete, under ACTH treatment, a large trimeric glycoprotein (CISP) presenting amino acid sequence similarity with thrombospondin-2. We also observed that the same cells synthesize and secrete thrombospondin-1 whereas under smaller amounts. The aim of this study was to investigate the regulation of these two secreted proteins by members of the TGF beta family of regulatory peptides. We developed an appropriate immunoprecipitation technique that allowed us to quantitate synthesis of thrombospondin-1 and CISP/thrombospondin-2 in a single assay. Using this assay, we observed that thrombospondin-1 and CISP/thrombospondin-2 syntheses were respectively stimulated threefold and twofold by a 24-h treatment with 2 ng/ml TGF beta 1. These inductions were dose-dependent (half-maximal effect: 0.2 ng/ml) and time-dependent (detectable after 5 h and plateauing between 15 and 25 h of treatment). They were not observed when transcription was blocked by RNA polymerase inhibitors such as 5,6-dichlorobenzimidazole riboside or actinomycin D. Among members of the TGF beta family, TGF beta 1 and TGF beta 2 and to a lesser extent activin could stimulate thrombospondin-1 and CISP/thrombospondin-2 synthesis, whereas inhibin and Müllerian inhibiting substance were inactive. Taken together, these data represent the first study on the regulation of both thrombospondin-1 and CISP/thrombospondin-2 by TGF betas. They further support the concept that TGF beta is a local regulator of adrenocortical functions.

Contribution of apoptosis to the phenotypic changes of adrenocortical cells in primary culture.

The aim of this study was to evaluate the occurrence and physiological consequences of apoptosis in primary cultures of bovine adrenocortical cells (of fasciculata-reticularis origin). Under ACTH-free culture conditions, we observed apoptotic cells in the cell layer and the accumulation of apoptotic bodies in the culture medium. These were hardly detectable in ACTH-supplemented cultures. Under ACTH-free conditions, the DNA content of apoptotic bodies collected over 48 h represented up to 10-15% of that of the cell layer at the onset of the culture (as compared to 3% in ACTH-supplemented cultures). Past the fourth day of culture in the absence of ACTh, most cells lacked several markers of their originating fasciculata-reticularis phenotype and progressively evolved to an undifferentiated phenotype. The vast majority of the apoptotic bodies released during the first 4 days of culture were immunoreactive for P450 17 alpha. Inversely, during the same period of time, the proliferating cells (PCNA-positive) did not appear to express P450 17 alpha. Therefore, apoptosis could contribute, together with dedifferentiation, to the phenotype shift observed in ACTH-depleted cultures of adrenal fasciculata-reticularis cells. These observations also characterize this endocrine cell system as an in vitro model for the study of hormone-repressed apoptosis.

Steroidogenic adrenocortical cells synthesize alpha 2-macroglobulin in vitro, not in vivo.

We previously identified alpha 2-macroglobulin as the major protein secreted by primary cultures of adrenocortical cells. We report here that in the adrenal gland, the distribution of alpha 2-macroglobulin in the adrenocortical tissue is restricted to the endothelium of blood vessels and that no immunoreactivity is found in steroidogenic cells. A time course study revealed that freshly dissociated bovine adrenocortical cells were void of alpha 2-macroglobulin immunoreactivity whereas the proportion of alpha 2-macroglobulin-positive cells reached more than two-thirds of the population between day 4 and day 7 of culture. Double immunoenzymatic labeling of 6-day-old cultures revealed a co-localization of alpha 2-macroglobulin and the steroidogenic enzyme P-450SCC. Treatment of 5-day-old cultures (expressing alpha 2-macroglobulin) for 24 h by either ACTH (10(-9)-10(-6) M) or alpha 2-macroglobulin (2.5 mg/ml) resulted in a marked decrease of the expression of alpha 2-macroglobulin. These data indicate that ACTH and plasmatic alpha 2-macroglobulin could physiologically repress alpha 2-macroglobulin expression in the adrenal cortex in vivo.

p53 immunolabeling in archival paraffin-embedded tissues: optimal protocol based on microwave heating for eight antibodies on lung carcinomas.

The prognostic value of p53 gene mutations is dealt with by several recent reports. However, retrospective assessment of p53 tumor status on archived samples has been prevented by p53 epitope alteration during routine fixation and embedding procedures. This study aimed at establishing a reproducible low-cost protocol to retrieve not only N-terminal, but also midregion and C-terminal, epitopes, with special attention to possible artifacts induced by epitope retrieval procedures. Using microwave heating, we compared the epitope retrieval efficiency of five solutions with eight commercial antibodies on 21 lung carcinomas for which frozen tissue and samples fixed with formalin and Bouin's liquid were available. All eight epitopes were retrieved, citrate buffer proving efficient for seven. PAb 240 epitope was restored by target unmasking fluid only. No false positivity was observed. Fixation-induced loss of p53 immunoreactivity was minimal for formalin (two of 10 tumors for one antibody each), more significant for Bouin (six of 10 tumors for one to five antibodies). On the other hand, staining intensity was maintained or even improved, and nonspecific staining reduced, through fixation. We conclude that p53 stabilization can be detected on routinely processed archival tumor samples with a reliability similar to that of frozen tissue by means of a microwave-based procedure and a panel of at least three antibodies, with epitopes on the N-terminal, C-terminal, and midpart of the molecule.