dimerizes with itself or with Bcl-2, promotes apoptosis when overproduced. In contrast, Mci-1, which was iso- lated as an early gene activated upon phorbol-ester ...
American Journal of Pathology, Vol. 149, No. 1, July 1996 Copynight © American Society for Investigative Pathologv
Immunodetection of Apoptosis-Regulating Proteins in Lymphomas from Patients with and without Human Immunodeficiency Virus Infection
Daniel Schlaifer,* Stanislaw Krajewski,t Sandrine Galoin,* Frangoise Rigal-Huguet,* Guy Laurent,* Patrice Massip,§ Jacques Pris,* Georges Delsol,$ John C. Reed,t and Pierre Brousset* From the Service d'Hematologie,* Clinique Dieulafoy, and the Laboratoire dAnatomie-Pathologique and CIGH/CNRS,t Centre Hospitalier Universitaire, H6pital de Purpan, Toulouse, France; the La Jolla Cancer Research Foundation,t and the Service de Maladies Infectieuses,s Centre Hospitalier de Rangueil, Toulouse, France
The expression of the apoptosis-regulating genes Bcl-2, Bcl-x, Bax, Mcl-i, and p53 was analyzed in 4 cases of human immunodeficiency virus (HIV)-associated Hodgkin's disease, in 36 cases of HIV-related non-Hodgkin's lymphomas (NHLs), and in 109 cases of nonHIV-related NHLs by using immunohistochemistry. HIV-associated Hodgkin's disease samples were positive for all markers. For the HIVrelated NHL samples, 36, 66, 88, 100, and 94% of the cases were Bcl-2, Bcl-x, Bax, Mdcl-, and p53 positive, respectively. For the HIV-unrelated NHLs, Bcl-2, Bcl-x, Bax, Mdcl-, and p53 were found to be expressed in 69, 65, 82, 83, and 42%, respectively. No significant differences were observed in Bax and Mcl-i staining between HIV-unrelated NHLs of B cell and Tcell types. In contrast, Bcl-2 was positive in 66/79 (83%) and 10/30 (33%) of B ceU and T ceU HIV-unrelated NHLs, respectively (P2 < 0.001). Peculiar patterns were observed for hairy ceU leukemia (Bax +, Bcl-2 +, Mcl-i -) and for anaplastic large ceU lymphoma (Bax +, Mdcl-i , Bcl2 ) in HIV-unrelated NHLs. Of interest, aU cases with a positive expression of Bax were also found to express either Mcl-i and/or Bcl-2, suggesting that Mcl-i and Bcl-2 may counteract the pro-apoptosis function of Bax in vivo by protein-protein interaction within
the tumor cell, as demonstrated previously in vitrob These results suggest that apoptosis regulation may have a role in the pathogenesis of some HIV-related and HIV-unrelated NHLs. (Am J Pathol 1996, 149:1 77-185)
Two major types of cell death have been described, namely necrosis (passive or accidental cell death) and apoptosis, a form of active cell suicide.1 Apoptosis is a morphologically stereotyped form of cell death, involving chromatin condensation and the formation of crescent-shaped deposits along the nuclear envelope. Of interest, apoptosis regulation has been shown to involve several genes, including Bcl-2 and Bc/-2-related genes.23 BcI-2 was discovered adjacent to the breakpoint of a chromosome rearrangement commonly seen in follicular B cell lymphoma, the t(14;18) translocation, and encodes for a 25-kd protein that is able to block apoptosis. Deregulated expression of Bcl-2 probably plays a central role in the development of follicular lymphoma, as a majority of cases contain the t(14;18) translocation.4 Furthermore, the Bcl-2 protein is expressed in -75% of nodal large cell lymphomas, whereas the t(14;18) translocation occurs in only 20 to 40% of the cases.4 Recently, several genes with Bc/-2-related sequences have been identified, including Bax,5 Mcl1,6 and BcI-x.7 The Bax protein, a Bcl-2-homologue that dimerizes with itself or with Bcl-2, promotes apoptosis when overproduced. In contrast, Mci-1, which was isolated as an early gene activated upon phorbol-esterSupported by the Lions Club Toulouse-Donjon, the Fondation pour la Recherche Medicale (SIDACTION), the American Cancer Society (DHP 32D), and the National Institutes of Health (CA-60181 and CA60421). Dr. Reed is a scholar of the Leukemia Society of America. Accepted for publication March 11, 1996. Address reprint requests to Dr. Pierre Brousset, Laboratoire d'Anatomie-Pathologique, Centre Hospitalier Universitaire, Hopital de Purpan, Place du Docteur Baylac, 31059 Toulouse Cedex, France. D. Schlaifer and P. Brousset contributed equally to this report.
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induced macrophage differentiation in the ML-1 human myeloid leukemia cell line, encodes for a 37-kd protein that is able to delay apoptosis.8 The Bcl-x gene was recently isolated by genomic and cDNA library screening by hybridization with a Bcl-2 probe7 and can generate at least two proteins, Bcl-x-L (long form), which is a blocker of apoptosis, and Bcl-x-S (short form), which promotes programmed cell death. We recently demonstrated that the Bcl-x gene product was detected in 86% of Hodgkin's disease tumor samples, suggesting that this gene could have a role in the pathogenesis of this lymphoma via apoptosis deregulation.9 The Bax gene is expressed in a broad range of tissues including lymph node, bone marrow, and spleen.5 Recently, human polyclonal antibodies were raised against Bax, McI-1, and Bcl-x, allowing for immunohistochemical distribution of these proteins in comparison with Bcl-2.10-12 In normal lymph nodes, Bax is widely expressed in lymphocytes and McI-1 intensively stains germinal center lymphocytes whereas Bcl-2 is present in only mantle zone and interfollicular lymphocytes.1011 Bcl-x immunostaining is observed in numerous cell types, including cortical thymocytes, activated lymphocytes, and plasma cells in reactive human lymph nodes.12 Mci-1 was also demonstrated to be expressed in t(14;18)-positive follicular lymphoma and in Hodgkin's disease (HD).10 This prompted us to study the immunoreactivity of Bax, Bcl-x, and Mci-1 in 40 cases of human immunodeficiency virus (HIV)related lymphomas and in 109 cases of HIV-unrelated non-Hodgkin's lymphoma (NHL) and to compare it with Bcl-2 expression and to the apoptosisregulating protein p5313 expression.
Materials and Methods Study Design, Patients' Profile, and in Situ Hybridization Lymphoid tissue was obtained from material submitted to the Pathology Department, Purpan's Hospital, Toulouse. There were 40 cases of HIV-related Iymphoma (4 cases of HD and 36 cases of HIV-NHL) occurring in HIV-seropositive patients in our files for the period 1990 to 1995 (Table 1). We have previously reported on the first 18 cases for Epstein-Barr virus (EBV) early RNAs (EBERs), latent membrane protein 1 (LMP-1), and Bcl-2 expression.14 Pathological material was accessible for in situ hybridization for all patients by using EBER oligonucleotides, as previously described.15 There were 109 cases of HIV-unrelated NHLs in our files available for this
study (Table 2). All tissues were lymph node biopsies that had been processed routinely, ie, fixed in ethanol-based Bouin's fluid (Duboscq-Brasil) and paraffin embedded. Subclassification of the malignant lymphomas was performed in accordance to the REAL classification.16 Immunohistochemistry for LMP-1, p53, Bcl-2, Bcl-x, Bax, and McI-1 was technically possible in all patients. No material was suitable for RNA or protein extraction. Statistical analysis was done by Yates' corrected x2 test. All tests were two sided.
Immunohistochemistty LMP-1 was detected as previously described.15 Bcl-2 monoclonal antibody was a gift from Dr. D. Y. Mason (Oxford, UK) and was used as reported.17 Bcl-x polyclonal antisera (1695) were raised against peptides corresponding to amino acids 46 to 66 of the human Bcl-x protein and were used as described previously.12 Mci-i polyclonal antibodies were raised against a peptide corresponding to amino acids 121 to 139 of the human Mci-1 protein and were used as previously described.10 Human Bax polyclonal antibodies (1712-12) were used as described previously.11 The working dilutions were 1:800 and 1:1000 for McI-1 and Bax, respectively. The monoclonal antibody DO-7 (M 7001, DAKO, France), which specifically detects human p53 (wildtype and mutant forms) and which is resistant to formalin fixation, was used.18 Antibodies were revealed using a streptavidin-biotin complex reagent (StreptABComplex/HRP Duet, DAKO; K692) on paraffin sections. Assessment for positivity was as follows: negative, 75% of tumor cells positive.9
Results
H/V-Positive Lymphomas Table 1 summarizes the characteristics of the HIVpositive tumors with regard to the histological subtypes according to the REAL classification,16 the immunophenotype, and the EBV status. As shown, 3/4 (75%) of the HIV-HDs were EBER and LMP-1 positive whereas 23/36 (63%) of the HIV-NHLs were EBER positive, as previously reported14'19 (Table 1 and data not shown). Bcl-2 and Bcl-x were detected in all four HIV-HD cases whereas Bax and Mci-1 were both detected in 3/4 (75%) of the cases. Among the 36 cases with HIV-NHL, 13 (36%) and 24 (66%) were found positive for Bcl-2 and Bcl-x stain-
Apoptosis Regulation in Lymphomas 179 AJPJuly 1996, Vol. 149, No. 1
Table 1. HIV-Related Lymphomas Case 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
Type HD HD HD HD M DL M DL DL
M DL DL M
EBER
DL DL DL DL DL DL DL DL DL BL BL BL
Bax ++ ++
+
++
_*
+ + +
++ ++ ++ + ++
+ ++ ++
M M BL
LMP-1
Immunohistochemistry Mci-1 Bcl-2
+
++
++ +
++
++
++ + ++ ++
+++ ++
++ +++ +++
+++ +++ +++ +++ +++ +++
++
+++
+++ +++
++
++
++
+++ +++ ++ ++
++ + ++
+
++ ++
++
++ +
+++
++
+++
+++ ++
++ ++ ++
M
++ ++ ++ ++ ++
+++ ++
++
+ ++ +++ ++ ++ ++ ++
++ ++
+ +
+++
M
DL
+++ +++
++ +
++ + ++
++
+ ++
++
++
++ + ++
++
M BL DL DL BL DL BL DL DL
p53
++
++
++
++ +++
Bcl-x
++ ++
+
+ + +
HD, Hodgkin's disease according to the REAL classification'6; DL, diffuse large B cell lymphoma; BL, Burkitt's-type lymphoma; M, nullcell-type anaplastic large cell lymphoma. All HIV-NHL cases, except for the M type, were of B cell origin as detected by a panel of monoclonal antibodies. *A few tumor cells positive (less than 1%).
ing, respectively (Table 1 and Figure 1). Positive internal controls for each case were the immunostaining of small reactive lymphocytes for Bcl-2 and of plasma cells for Bcl-x. Bcl-x staining was similar to Bcl-2 in general but stronger in the HIV-HD cases (Table 1). Bax immunostaining was found positive in the cytoplasm of the tumor cells in 32/36 (88%) of the cases (Figure 2) whereas McI-1 stained in 100% of the tumor samples (Figure 3). Positive internal controls for both Bax and McI-1 were interfollicular and germinal center lymphocytes. Nuclear p53 immunostaining was found in 4/4 and 34/36 (94%) of, respectively, HIV-HD and HIV-NHL cases (Table 1). Interestingly, in some cases, phantom cells within necrotic areas were found to be strongly labeled with
anti-p53 antibodies compared with viable cells. Furthermore, both mitotic and interphasic cells were found to express p53 (data not shown). Bcl-2, Bcl-x, Bax, Mci-1, and p53 immunodetection were compared with EBV status. LMP-1 staining positively correlated with Bcl-2 positivity (P < 0.05), in line with the possible up-regulation of Bcl-2 by the LMP-1 gene.20 Furthermore, 21/26 (80%) of the cases were both EBV positive and Bcl-x positive and 7/14 (50%) were both EBV negative and Bcl-x negative (P = 0.070). No significant relation was found between EBV positivity and Bax, Mci-1, and p53 staining. Dual expression of Bcl-2/p53, Bax/p53, Bcl-x/p53, and McI-1/ p53 was 17/40 (42%), 35/40 (87%), 28/40 (70%), and 37/40 (92%), respectively.
180 Schlaifer et al AJPJuly 1996, Vol. 149, No. 1
Table 2. Non-HIV-Related Lymphomas Lymphoid neoplasms $
Bax
Mci-1
Bcl-2
Bcl-x
p53
+
+++
B-cell neoplasms Precursor B cell neoplasms Precursor B-lymphoblastic leukemia 1
2 Peripheral B cell neoplasms B cell chronic lymphocytic leukemia 3 CLL 4 CLL 5 CLL Lymphoplasmacytoid lymphoma 6 7 8 9 Mantle cell lymphoma 10 11 12 13 14 15 16 17 Follicular center cell lymphomat 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 Marginal zone B-cell lymphoma MALT lymphoma 44 45 46 B monocytoid 47 48 49 Splenic marginal zone lymphoma 50 51 Hairy cell leukemia 52 53 54
+
+++
-
++
++
+++
-+++
+ +++ ++
++ +
+++ +++ +++
-+++
-
+++
++ +
+
+++
++
+ +++
-
+
+ +
_
+
++ 0 ++
-
+++-
-
+
-
+++++ +3 +++ +
-
+
+++
+
++
++
-
+
6+ -
+
++
-
-
+ + + + + ++ ++ ++ ++
++ +
++
+++
++ ++
-*
++ + ++
+ +
*
+++ +++
-
+ + + + + + + +
+++ +
+ + +
+ + +
+
-
++ +
+++ +++
++
-
+ ++
+
-
+
+++
++
++
-
+
++
++
+++
+ ++ +
++
+++
++
-
+ ++ +
+ ++
++
+
-
+
+
++
* *
+ + + ++ ++ + +
+ -
++
+++-
++ ++
+++ -
*
++++
+
+
++
+
++
-
+-
-
++
+++
-
+
+
_
++
++
++
-
-
++-
-
+
+
+
-
+
+-++ +
+
-
+
++
-
-
+
-
Apoptosis Regulation in Lymphomas 181 AJPJuly 1996, Vol. 149, No. 1
Table 2. Continued
Lymphoid neoplasms $
Diffuse large B cell lymphoma 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 + 76 T-cell-rich B cell lymphoma 77 T-cell-rich B cell lymphoma + Burkitt's lymphoma 78 79 T cell neoplasms Precursor T cell neoplasms Precursor T-lymphoblastic leukemia 80 81 Peripheral T cell neoplasms T cell chronic lymphocytic leukemia 82 Large granular lymphocyte leukemia 83 84
Bax
Mci-1
Bcl-2
+ ++ ++ + ++ +++
++ +
Bcl-x
p53
+++
+
+++
++
+
+ +++ +++ +++ +++++ + ++ +
+ ++
-+++ -+++
+++ + +
+
+ ++ ++ + ++ ++ ++ +++ + +++ ++
-
++ + +++ +++ + ++ + + ++ +
+ +
++
++ +
-+++ +++-* ++ + ++
+++-
+
-
++
+
+
++-
+ + ++ + +++ ++ + ++ ++ +++ +
+ +
++
+
++ ++ ++ +
+ + + + + +
++ ++
+ +
+
ND
ND
+
++
+++-
+
++
-
+
++
+
+ + ++
++ + +
+ +
+ +
+
+
Mycosis fungoides 85 86 87
-+++
Peripheral T cell lymphomas (unspecified) 88 89 90
91 92 93 94 95 96 97 Angioimmunoblastic T cell lymphoma 98 99 Anaplastic large cell lymphomat 100 101 102 103 104 105 106 107 108 109
+ +++ ++ -
+ ++
+++ +
++ + ++
-
+++
+ ++ + + +++ ++
+
+ ++ ++
++ + ++ ++ ++ +
+++ ++
-
-
-
+ + + ++ +
-
+
-
+ + +
-
++
+
+
+++
++
+ +
-
-
++ ++ ++ + ++ -++ +++ ++ + +++ ++ +++ +++-
+
Neoplasms were classified according to the REAL classification. 16 *A few cells positive (less than 1%).
+ +
tt(14;18) as determined by PCR; not known, cases 18 to 26; positive, cases 27 to 33; negative, cases 34 to 43. tt(2;5) as determined by PCR; not known, cases 100 and 101; negative, cases 102 and 103; positive, cases 104 to 109.
+ +
+
+ +
+
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Figure 1. Heterogenieous cytoplasmic staining observed with antiBcl-x antibody in a case ofAIDS-NHL of immunoblastic type. Virtually all tumor cells are labeled. Paraffin section, peroxidase-diaminobenzidine; original magnification, x 1000.
Figure 2. Strong granular and dot-like staining obtained with antiBax antibody in the cytoplasm of a case of AIDS-NHL of centroblasticimmunoblastic type. Paraffin section, peroxidase-diaminobenzidine; original magnification, X 800.
Figure 3. Strong cytoplasmic labeling observed with anti-Mcl-1 antibody in a case of AIDS-NHL of immunoblastic type. Note that all cells display an intense staining. Paraffin section, peroxidase-diaminobenzidine; original magnification, X 800.
Figure 4. Immunostaining observed with anti-Bax antisera in a case oflarge B cell lymphoma. Paraffin section, peroxidase-diaminobenzidine; original magnification, X500.
H/V-Negative Lymphomas Bax, Mci-1, Bcl-x, and Bcl-2 protein expression was evaluated in 109 NHLs with specific polyclonal (Bax, Bcl-x, & Mci-1) and monoclonal (Bcl-2) antibodies that do not cross-react1011 (Table 2). Normal and reactive lymph nodes were also examined to allow a comparison with the malignant conditions. In normal and reactive lymph nodes, Bax was widely expressed in germinal center and interfollicular lymphocytes, Mci-1 and Bcl-2 stained germinal center and mantle zone lymphocytes, respectively, and Bcl-x stained plasma cells and large cells within the interfollicular area, as previously shown10-12 (data not shown). In malignant lymph nodes, Bax reactivity was observed in the cytoplasm of the tumor cells in 90/109 (82%) of the NHL samples (Table 2 and Figure 4). No significant differences in Bax positivity were observed between B phenotype (68/79 or 86%) and T phenotype (22/30 or 73%) cases. McI-1 was detected in 91/109 (83%) of the cases (Figure 5)
I
I Figure 5. Immunostaining observed in a case of peripheral T cell Iymphoma uith anti-Mcl-1 antisera. Paraffin section, peroxidase-diaminobenzidine; originial magnification, X 500.
and, of note, was absent in the tumor cells of 3/3 hairy cell leukemia samples and of 4/8 mantle cell lymphoma cases (Table 2). Of the T cell and B cell lymphoma cases, 26/30 (86%) and 65/79 (82%) were Mci-1 positive, respectively (not significant).
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Bcl-2 stained 76/109 (69%) of the cases, and low grade as well as high grade B cell lymphomas were frequently Bcl-2 positive, as previously reported.17 Of interest, we found that all 10 cases of anaplastic large cell lymphomas were Bcl-2 negative (Table 2). No significant differences were observed in the t(14; 18)-positive and -negative follicular lymphoma samples regarding Bax, Mci-1, or Bcl-2 staining (Table 2). In contrast, a significant difference in the Bcl-2 positivity was observed between the B cell (66/79 or 83%) and the T cell (10/30 or 33%) NHLs (P < 0.001). All Bax-positive cases were either Bcl-2 and/or Mci-1 positive. Bcl-x staining was found in 71/108 (65%) of the cases; no significant difference was found between B cell (52/79 or 65%) or T cell (19/29 or 65%) NHLs (Table 2). p53 reactivity was observed in 46/108 (42%) of the cases without significant difference between B and T cell subtype NHLs (Table 2).
Discussion In this report, we have described for the first time the expression of several different apoptosis-regulating genes in HIV-related and HIV-unrelated lymphomas. Bcl-2 is the founding member of an expanding family of proteins, including Bax,5 Bcl-x,7 and McI-l.6 Bcl-2 has an anti-apoptotic function by heterodimerizing with Bax.5 Overproduction of Bax is able to promote apoptosis. Bax by itself probably cannot induce cell death but requires a cell death signal.21 Bcl-x is another Bcl-2 homologue, which in its long form is able to block apoptosis whereas in its short form favors cell death.7 We were able to detect Bcl-2 expression in 42% of HIV-related lymphomas, in line with our previous findings.14 Of interest, Bcl-x was highly expressed in 100% of the HIV-HD cases, as previously found in HIV-unrelated HD,9 suggesting that Bcl-x may have a role in the pathogenesis of HD. We also found that 66% of the HIV-NHL cases and 65% of the non-HIV-related NHLs were positive for Bcl-x expression. Polyclonal antibodies used are unable to differentiate the Bcl-x-L and Bcl-x-S forms. However, a recent work suggests that Bcl-x-L is the most frequently expressed isoform within NHLs,22 as has been previously shown in normal tissue.7 Other apoptosis-regulating genes like Bax and Mcl-1 were highly expressed in the tumor cells of HIV-related and HIV-unrelated lymphomas, and of interest, all Bax-positive tumors were found to express either Bcl-2 or Mci-1 anti-apoptosis protein(s), strongly suggesting that the presence of the apoptosisinducing protein Bax within the tumor cells must be
associated with apoptosis-inhibiting proteins for allowing malignant cell survival. Of interest, there was a contrast between the normal patterns of Bcl-2, Bcl-x, Mci-1, and Bax in normal lymph nodes and in NHL; normal germinal center lymphocytes usually express Mci-1 and Bax, just as most of the NHLs tested here, whereas normal germinal center cells usually do not express Bcl-2 or Bcl-x.23 Thus, the Bcl-2 and Bcl-x expression seen in HIV-related immunoblastic B cell lymphomas and in HIV-unrelated large B cell lymphomas is abnormal relative to the normal counterparts of these B cells. Of note, we found that Bcl-2 expression was significantly less frequent in HIV-negative NHLs of T cell origin when compared with B cell NHLs. Furthermore, the consistent lack of detection of Bcl-2 within HIV-unrelated anaplastic large cell lymphoma samples and the frequent expression of Bax and Mci-1 may be of diagnostic value in differentiating HD from anaplastic large cell lymphoma. Wild-type p53 has been shown to negatively regulate cell growth and to halt the proliferation of DNAdamaged cells in the G1 phase of the cell cycle, allowing them to repair DNA damage before S phase or to go into apoptotic cell death.24 In contrast, mutations of the p53 gene appear to be oncogeniC,24 and mutated p53 was found in numerous human cancer types including lymphomas. 13 Mutated p53 has been found in 10 to 16% of HIV-NHL samples, whereas p53 was detected by immunohistochemistry in 7/11 (63%) of the cases in one study.25 The types and incidence of the p53 mutations in HIV-NHL have not been found to differ from those found in HIV-unrelated NHL.25 We found a high expression of the p53 nuclear phosphoprotein in HIV-HD (100%) and in HIV-NHL (94%) samples, similar to Nakamura et al.25 The precise role of p53 overexpression in HIV-related lymphoma pathogenesis remains to be determined as immunohistochemical detection of p53 has not been found to correlate with p53 mutations in NHL.13 As previously reported for HD, p53 detection was not correlated in our study with EBV presence within the tumor cells. Dual expression of Bcl-2 and p53 was detected in 17/40 (42%) of the cases, and no correlation was found between these two markers, suggesting that AIDS-related lymphomas may have a different cell death pathway regulation when compared with colorectal26 or breast carcinomas.27 Nevertheless, we found that 87% of the HIV-related NHL cases co-expressed Bax and p53, of interest as it has been reported that p53 is a direct transcriptional activator of Bax.28 The finding of a strong p53 staining in mitotic cells questions the ability of this protein to block the cell cycle in lym-
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phoma cells, suggesting that either p53 is mutated and non functional or that the tumor cells have another abnormality in their cell cycle regulation. Furthermore, the high expression of p53 in necrotic cells may indicate that, at least in some cases, the form of p53 protein detected here could play a role in inducing cell death. In conclusion, we demonstrated that the apoptosis-regulating genes Bcl-2, Bcl-x, Bax, Mcl-i, and p53 are highly expressed in HIV-HD and HIV-NHL or HIV-unrelated NHL patients, strongly suggesting that programmed cell death deregulation may be involved in lymphoma pathogenesis. Furthermore, in the HIV-NHLs, EBV presence in the tumor cells was found to correlate with the high level of expression of the apoptosis-regulating proteins Bcl-2 and Bcl-x. Other recently described apoptosis-regulating proteins29-32 may be further investigated in the future.
Acknowledgments We thank Michel March for expert technical assistance and Dr. Catherine Payen for statistical analysis of the data.
References 1. Wyllie AH, Kerr JFR, Currie AR: Cell death: the significance of apoptosis. Int Rev Cytol 1980, 68:251-306 2. Reed JC: Bcl-2 and the regulation of programmed cell death. J Cell Biol 1994, 124:1-6 3. Williams GT, Smith CA: Molecular regulation of apoptosis: genetic controls on cell death. Cell 1993, 74:777-779 4. Pezzella F, Gatter K: What is the value of Bcl-2 protein
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detection for histopathologists? Histopathology 1995, 26:89-93 Oltvai ZN, Milliman CL, Korsmeyer SJ: Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 1993, 74:609-619 Kozopas KM, Yang T, Buchan HL, Zhou P, Craig RW: MCL1, a gene expressed in programmed myeloid cell differentiation, has sequence similarity to BCL2. Proc Natl Acad Sci USA 1993, 90:3516-3520 Boise LH, Gonzalez-Garcia M, Postema CE, Ding L, Lindsten T, Turka LA, Mao X, Nunez G, Thompson CB: Bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell 1993, 74:597608 Reynolds JE, Yang T, Qian L, Jenkinson JD, Zhou P, Eastman A, Craig RW: Mci-1, a member of the Bcl-2 family, delays apoptosis induced by c-myc overexpression in Chinese hamster ovary cells. Cancer Res 1994, 54:6348-6352
9. Schlaifer D, March M, Krajewski S, Laurent G, Pris J, Delsol G, Reed JC, Brousset P: High expression of the bcl-x gene in Reed-Sternberg cells of Hodgkin's disease. Blood 1995, 85:2671-2674 10. Krajewski S, Bodrug S, Gascoyne R, Berean K, Krajewska M, Reed JC: Immunohistochemical analysis of Mci-1 and bcl-2 proteins in normal and neoplastic lymph nodes. Am J Pathol 1994, 145:515-525 11. Krajewski S, Blomqvist C, Franssila K, Krajewska M, Wasenius V-M, Niskanen E, Nordling S, Reed JC: Reduced expression of pro-apoptotic gene Bax is associated with poor response rates to combination chemotherapy and shorter survival in women with metastasis breast adenocarcinomas. Cancer Res 1995, 55:4471-4478 12. Krajewski S, Krajewska M, Shabaik A, Wang HG, Irie S, Fong L, Reed JC: Immunohistochemical analysis of in vivo patterns of bcl-x expression. Cancer Res 1994, 54:5501-5507 13. Imamura J, Miyoshi I, Koeffler HP: P53 in hematologic malignancies. Blood 1994, 84:2412-2421 14. Schlaifer D, Brousset P, Attal M, Massip P, Payen C, Marchou B, Huguet F, Muller C, Laurent G, Pris J, Delsol G: Bcl-2 proto-oncogene and Epstein-Barr virus latent membrane protein-1 expression in AIDS-related lymphoma. Histopathology 1994, 25:77-82 15. Brousset P, Butet V, Chittal S, Selves J, Delsol G: Comparison of in situ hybridization using different nonisotopic probes for detection of Epstein-Barr virus in nasopharyngeal carcinoma and immunohistochemical correlation with anti-latent membrane protein antibody. Lab Invest 1992, 67:457-464 16. Harris NL, Jaffe ES, Stein H, Banks PM, Chan JKC, Cleary ML, Delsol G, De Wolf-Peeters C, Falini B, Gatter KC, Grogan TM, Isaacson PG, Knowles DM, Mason DY, Muller-Hermelink HK, Pileri SA, Piris MA, Ralfkiaer E, Warnke RA: A revised European-American classification of lymphoid neoplasms: a proposal from the International Lymphoma Study Group. Blood 1994, 84: 1361-1 392 17. Pezzella F, Tse AGD, Cordell JL, Pulford KAF, Gatter KC, Mason DY: Expression of the bcl-2 oncogene protein is not specific for the 14;18 chromosomal translocation. Am J Pathol 1990, 137:225-232 18. Vojtesek B, Bartek J, Midgley CA, Lane DP: An immunohistochemical analysis of the human nuclear phosphoprotein p53: new monoclonal antibodies and epitope mapping using recombinant p53. J Immunol Methods 1992, 151:237-244 19. Hamilton-Dutoit SJ, Raphael M, Audouin J, Diebold J, Lisse I, Pedersen C, Oksenhendler E, Marelle L, Pallesen G: In situ demonstration of Epstein-Barr virus small RNAs (EBER1) in acquired immunodeficiency syndrome-related lymphomas: correlation with tumor morphology and primary site. Blood 1993, 82:619-624 20. Henderson S, Rowe M, Gregory C, Croom-Carter D, Wang F, Longnecker R, Kieff E, Rickinson A: Induction
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21. 22.
23.
24.
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