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2.
Leukemia ; 29(11): 2162-72, 2015 Nov.
Article in English | MEDLINE | ID: mdl-25987255

ABSTRACT

Anaplastic lymphoma kinase-positive (ALK+) anaplastic large-cell lymphoma (ALCL) is an aggressive T-cell non-Hodgkin lymphoma characterized by the t(2;5), resulting in the overexpression of nucleophosmin (NPM)-ALK, which is known to activate the phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway, resulting in cell cycle and apoptosis deregulation. ALK+ ALCL is also characterized by strong activator protein-1 (AP-1) activity and overexpression of two AP-1 transcription factors, CJUN and JUNB. Here, we hypothesized that a biologic link between AP-1 and AKT kinase may exist, thus contributing to ALCL oncogenesis. We show that JUNB and CJUN bind directly to the AKT1 promoter, inducing AKT1 transcription in ALK+ ALCL. Knockdown of JUNB and CJUN in ALK+ ALCL cell lines downregulated AKT1 mRNA and promoter activity and was associated with lower AKT1 protein expression and activation. We provide evidence that this is a transcriptional control mechanism shared by other cell types even though it may operate in a way that is cell context-specific. In addition, STAT3 (signal transducer and activator of transcription 3)-induced control of AKT1 transcription was functional in ALK+ ALCL and blocking of STAT3 and AP-1 signaling synergistically affected cell proliferation and colony formation. Our findings uncover a novel transcriptional crosstalk mechanism that links AP-1 and AKT kinase, which coordinate uncontrolled cell proliferation and survival in ALK+ ALCL.


Subject(s)
Gene Expression Regulation, Neoplastic , Lymphoma, Large-Cell, Anaplastic/genetics , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-jun/physiology , Receptor Protein-Tyrosine Kinases/analysis , Transcription Factors/physiology , Anaplastic Lymphoma Kinase , Cell Line, Tumor , Humans , Promoter Regions, Genetic , STAT3 Transcription Factor/physiology , Transcription Factor AP-1/physiology
3.
Leukemia ; 25(5): 856-67, 2011 May.
Article in English | MEDLINE | ID: mdl-21394100

ABSTRACT

p53 is frequently wild type (wt) in diffuse large B-cell lymphoma (DLBCL) associated with t(14;18)(q32;q21) that overexpresses BCL2. Nutlin-3a is a small molecule that activates the p53 pathway by disrupting p53-MDM2 interaction. We show that nutlin-3a activates p53 in DLBCL cells associated with t(14;18)(q32;q21), BCL2 overexpression and wt p53, resulting in cell cycle arrest and apoptosis. Nutlin-3a treatment had similar effects on DLBCL cells of activated B-cell phenotype with wt p53. Cell cycle arrest was associated with upregulation of p21. Nutlin-3a-induced apoptosis was accompanied by BAX and PUMA upregulation, BCL-XL downregulation, serine-70 dephosphorylation of BCL2, direct binding of BCL2 by p53, caspase-9 upregulation and caspase-3 cleavage. Cell death was reduced when p53-dependent transactivation activity was inhibited by pifithrin-α (PFT-α), or PFT-µ inhibited direct p53 targeting of mitochondria. Nutlin-3a sensitized activation of the intrinsic apoptotic pathway by BCL2 inhibitors in t(14;18)-positive DLBCL cells with wt p53, and enhanced doxorubicin cytotoxicity against t(14;18)-positive DLBCL cells with wt or mutant p53, the latter in part via p73 upregulation. Nutlin-3a treatment in a xenograft animal lymphoma model inhibited growth of t(14;18)-positive DLBCL tumors, associated with increased apoptosis and decreased proliferation. These data suggest that disruption of the p53-MDM2 interaction by nutlin-3a offers a novel therapeutic approach for DLBCL associated with t(14;18)(q32;q21).


Subject(s)
Chromosomes, Human, Pair 14/genetics , Chromosomes, Human, Pair 18/genetics , Imidazoles/pharmacology , Lymphoma, Large B-Cell, Diffuse/metabolism , Piperazines/pharmacology , Proto-Oncogene Proteins c-bcl-2/metabolism , Proto-Oncogene Proteins c-mdm2/antagonists & inhibitors , Translocation, Genetic/genetics , Tumor Suppressor Protein p53/metabolism , Animals , Antineoplastic Agents/pharmacology , Apoptosis , Blotting, Western , Cell Cycle , Cell Proliferation , Disease Models, Animal , Female , Fluorescent Antibody Technique , Humans , Immunoprecipitation , Lymphoma, Large B-Cell, Diffuse/drug therapy , Lymphoma, Large B-Cell, Diffuse/genetics , Mice , Mice, SCID , Mutation/genetics , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-mdm2/genetics , Proto-Oncogene Proteins c-mdm2/metabolism , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Tumor Cells, Cultured , Tumor Suppressor Protein p53/genetics , Xenograft Model Antitumor Assays
4.
Oncogene ; 29(46): 6125-37, 2010 Nov 18.
Article in English | MEDLINE | ID: mdl-20802511

ABSTRACT

Jun activation domain-binding protein 1 (JAB1) is a multifunctional protein that participates in the control of cell proliferation and the stability of multiple proteins. JAB1 overexpression has been implicated in the pathogenesis of human cancer. JAB1 regulates several key proteins and thereby produces varied effects on cell cycle progression, genome stability and cell survival. However, the biological significance of JAB1 activity in these cellular signaling pathways is unclear. Therefore, we developed mice that were deficient in Jab1 and analyzed the null embryos and heterozygous cells. This disruption of Jab1 in mice resulted in early embryonic lethality due to accelerated apoptosis. Loss of Jab1 expression sensitized both mouse primary embryonic fibroblasts and osteosarcoma cells to γ-radiation-induced apoptosis, with an increase in spontaneous DNA damage and homologous recombination (HR) defects, both of which correlated with reduced levels of the DNA repair protein Rad51 and elevated levels of p53. Furthermore, the accumulated p53 directly binds to Rad51 promoter, inhibits its activity and represents a major mechanism underlying the HR repair defect in Jab1-deficient cells. These results indicate that Jab1 is essential for efficient DNA repair and mechanistically link Jab1 to the maintenance of genome integrity and to cell survival.


Subject(s)
DNA Damage , DNA Repair , Intracellular Signaling Peptides and Proteins/physiology , Peptide Hydrolases/physiology , Animals , Apoptosis , Blastocyst/cytology , COP9 Signalosome Complex , Cell Proliferation , Cell Survival , Embryonic Development , Intracellular Signaling Peptides and Proteins/analysis , Mice , Peptide Hydrolases/analysis , Rad51 Recombinase/physiology , Tumor Suppressor Protein p53/physiology
5.
Leukemia ; 23(12): 2290-9, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19741726

ABSTRACT

p53 is expressed frequently, but is rarely mutated in anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) tumours. Nutlin-3a is a recently developed small molecule that targets Mdm2, a critical negative regulator of p53, and disrupts the p53-Mdm2 interaction resulting in p53 stabilization and activation. We show that nutlin-3a activates p53 in ALK+ ALCL cells carrying a wild type (wt) or mutated but partially functional p53 gene resulting in p53-dependent cell-cycle arrest and apoptosis. Cell-cycle arrest was associated with upregulation of the cyclin-dependent kinase inhibitor p21. Nutlin-3a-induced apoptotic cell death was accompanied by Bax and Puma upregulation, downregulation of Bcl-xl, survivin, and caspase-3 cleavage, and this was reduced when p53-dependent transactivation activity was inhibited by pifithrin-alpha, or when pifithrin-mu was used to inhibit direct p53 targeting of mitochondria. Nutlin-3a sensitized the activation of the extrinsic apoptotic pathway in wt-p53 ALK+ ALCL cells, in part, through upregulation of DR-5 and downregulation of c-Flip(S/L), and was synergistic with TRAIL in cell death induction. In addition, nutlin-3a treatment enhanced doxorubicin cytotoxicity against ALK+ ALCL cells harbouring mt p53, and this was associated with p73 upregulation. These data suggest that disruption of the p53-mdm2 interaction by nutlin-3a offers a novel therapeutic approach for ALK+ ALCL patients.


Subject(s)
Imidazoles/pharmacology , Lymphoma, Large-Cell, Anaplastic/drug therapy , Mutation , Piperazines/pharmacology , Tumor Suppressor Protein p53/drug effects , Apoptosis , Apoptosis Regulatory Proteins/biosynthesis , Cell Cycle , Cell Line, Tumor , Doxorubicin/pharmacology , Drug Synergism , Humans , Imidazoles/therapeutic use , Lymphoma, Large-Cell, Anaplastic/genetics , Piperazines/therapeutic use , TNF-Related Apoptosis-Inducing Ligand/pharmacology , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism
6.
Leukemia ; 23(4): 784-90, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19225536

ABSTRACT

Mantle cell lymphoma (MCL) is a clinically aggressive B-cell non-Hodgkin lymphoma characterized by the t(11;14)(q13;q32) and overexpression of cyclin D1. A high proportion of MCL tumors harbor wild-type (wt) and potentially functional p53 gene. We show here that stabilization and activation of wt-p53 using a recently developed potent MDM2 inhibitor, nutlin 3A, results in significant p53-dependent G1-S cell cycle arrest and apoptosis in MCL cells through regulation of p53 target genes. As mTOR signaling is activated in MCL and may control cyclin D1 levels, we show that p53 activation may downregulate the AKT/mTOR pathway through a mechanism involving AMP kinase (AMPK). Despite the non-genotoxic mode of nutlin 3A treatment, we show evidence that stabilization of p53 is associated with its phosphorylation at serine 15 residue and activation of AMPK. Stimulation of AMPK kinase activity using AICAR inhibits phosphorylation of critical downstream effectors of mTOR signaling, such as 4E-BP1 and rpS6. Pharmacologic inhibition of AMPK using compound C in nutlin-3A-treated MCL cells harboring wt-p53 did not affect the level of (ser15)p-p53, suggesting that the (ser15)p-p53 --> AMPK is the direction involved in the p53/AMPK/mTOR cross talk. These data establish a p53 --> AMPK --> mTOR mechanism in MCL and uncover a novel biologic effect of potent MDM2 inhibitors in preclinical models of MCL.


Subject(s)
Lymphoma, Mantle-Cell/metabolism , Protein Kinases/metabolism , Tumor Suppressor Protein p53/metabolism , AMP-Activated Protein Kinase Kinases , Apoptosis , Down-Regulation , Imidazoles/pharmacology , Phosphorylation , Piperazines/pharmacology , Protein Stability , Proto-Oncogene Proteins c-mdm2/antagonists & inhibitors , Receptor Cross-Talk , Signal Transduction , TOR Serine-Threonine Kinases , Tumor Cells, Cultured
7.
Leukemia ; 19(9): 1663-9, 2005 Sep.
Article in English | MEDLINE | ID: mdl-15990866

ABSTRACT

Anaplastic large-cell lymphoma (ALCL), as defined in the World Health Organization, is a heterogeneous category in which a subset of cases is associated with the t(2;5)(p23;q35) or variant translocations resulting in overexpression of anaplastic lymphoma kinase (ALK). p53 has not been assessed in currently defined subsets of ALCL tumors. In this study, we assessed ALK+ and ALK- ALCL tumors for p53 gene alterations using PCR, single-strand conformation polymorphism and direct sequencing methods. We also immunohistochemically assessed ALCL tumors for p53 expression. Three of 36 (8%) ALCL tumors (1/14 ALK+, 2/22 ALK-) with adequate DNA showed p53 gene mutations. By contrast, p53 was overexpressed in 36 of 55 (65%) ALCL tumors (16 ALK+, 20 ALK-). p21, a target of p53, was expressed in 15 of 31 (48%) ALCL tumors including seven of 15 (47%) p53-positive tumors. p21 expression in a subset of ALCL suggests the presence of functional p53 protein. Apoptotic rate was significantly higher in p53-positive than p53-negative tumors (mean 2.78 vs 0.91%, P = 0.0003). We conclude that the p53 gene is rarely mutated in ALK+ and ALK- ALCL tumors. Nevertheless, wild-type p53 gene product is commonly overexpressed in ALCL and may be functional in a subset of these tumors.


Subject(s)
Gene Expression Regulation, Neoplastic , Genes, p53/genetics , Lymphoma, Large B-Cell, Diffuse/genetics , Protein-Tyrosine Kinases/genetics , Tumor Suppressor Protein p53/biosynthesis , Tumor Suppressor Protein p53/genetics , Adult , Anaplastic Lymphoma Kinase , Apoptosis/physiology , Cell Cycle Proteins/biosynthesis , Cell Cycle Proteins/genetics , Cell Proliferation , Cloning, Molecular , Cyclin-Dependent Kinase Inhibitor p21 , Humans , Lymphoma, Large B-Cell, Diffuse/metabolism , Lymphoma, Large B-Cell, Diffuse/pathology , Middle Aged , Mutation , Nuclear Proteins/biosynthesis , Nuclear Proteins/genetics , Polymerase Chain Reaction , Proto-Oncogene Proteins/biosynthesis , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-mdm2 , Receptor Protein-Tyrosine Kinases , Sequence Analysis, DNA/methods
8.
Leukemia ; 18(11): 1872-8, 2004 Nov.
Article in English | MEDLINE | ID: mdl-15385932

ABSTRACT

Using a cDNA microarray, we found that suppressor of cytokine signaling 3 (SOCS3) is highly expressed in anaplastic lymphoma kinase (ALK)+ anaplastic large cell lymphoma (ALCL) cell lines. As SOCS3 is induced by activated signal transducer and activator of transcription 3 (STAT3), and ALK activates STAT3, we hypothesized that SOCS3 may play a role in ALK+ ALCL pathogenesis via the Janus kinase 3 (JAK3)-STAT3 pathway. Using ALCL cell lines, we show by coimmunoprecipitation experiments that SOCS3 physically binds with JAK3 in vitro, and that JAK3 inhibition by WHI-P154 downregulates SOCS3 expression. Western blot analysis confirmed expression of SOCS3 and also showed coexpression of phosphorylated (activated) STAT3 (pSTAT3). Direct sequencing of the SOCS3 gene showed no mutations or alternative splicing. In ALCL tumors that were assessed by immunohistochemistry, nine of 12 (75%) ALK+ tumors were SOCS3 positive and eight (67%) coexpressed pSTAT3. In comparison, 18 of 25 (72%) ALK-- tumors were SOCS3 positive and seven (28%) coexpressed pSTAT3. These results show that SOCS3 is overexpressed in ALCL, attributable to JAK3-STAT3 activation and likely related to ALK in ALK+ tumors. However, SOCS3 is also expressed in tumors that lack STAT3 and ALK suggesting alternative mechanisms of upregulation.


Subject(s)
DNA-Binding Proteins/metabolism , Gene Expression Regulation, Neoplastic , Lymphoma, Large-Cell, Anaplastic/metabolism , Protein-Tyrosine Kinases/metabolism , Repressor Proteins/metabolism , Signal Transduction , Trans-Activators/metabolism , Transcription Factors/metabolism , Alternative Splicing , Anaplastic Lymphoma Kinase , Gene Expression Profiling , Humans , Immunoprecipitation , Janus Kinase 3 , Lymphoma, Large-Cell, Anaplastic/pathology , Mutation , Oligonucleotide Array Sequence Analysis , Phosphorylation , Protein-Tyrosine Kinases/antagonists & inhibitors , Quinazolines/pharmacology , Receptor Protein-Tyrosine Kinases , Repressor Proteins/genetics , STAT3 Transcription Factor , Suppressor of Cytokine Signaling 3 Protein , Suppressor of Cytokine Signaling Proteins , Transcription Factors/genetics , Tumor Cells, Cultured
9.
J Clin Oncol ; 20(12): 2876-80, 2002 Jun 15.
Article in English | MEDLINE | ID: mdl-12065565

ABSTRACT

PURPOSE: Methotrexate (MTX) is active against lymphomas, but transport or polyglutamylation mutations confer MTX resistance. Because trimetrexate (TMTX) enters cells by passive diffusion and is not polyglutamylated, its activity in relapsed T-cell lymphoma was investigated. PATIENTS AND METHODS: Eligible patients had histologically confirmed relapsed T-cell lymphoma involving the skin, had received more than one previous regimen, were older than 16 years, had normal organ function, and had no CNS disease or serious infections, including human immunodeficiency virus. TMTX (200 mg/m(2)) was given intravenously every 14 days without topical or systemic corticosteroids. Patients who responded received up to 12 doses. RESULTS: Twenty patients were assessable for response. Median age was 59 years (range, 45 to 87 years); 13 patients were men. Three patients had anaplastic large-cell lymphoma, 15 had mycosis fungoides or Sézary syndrome (14 with large-cell transformation), and two had peripheral T-cell lymphoma. Serum lactate dehydrogenase was high in 35%, and beta-2 microglobulin was more than 3.0 mg/L in 35% of patients. The median number of previous regimens was three (range, two to 15) and included MTX in five patients. Disease was refractory to the regimen immediately preceding TMTX in 85% of patients. Responses were complete in one and partial in eight patients (overall response rate, 45%). Two of five patients previously treated with MTX responded. Grade 3 or 4 mucositis was observed after 4%, infection after 3%, neutropenic fever after 6%, neutrophils less than 100/microL after 4%, and platelets less than 10,000/microL after 3% of TMTX doses. CONCLUSION: TMTX is active with acceptable toxicity in this population and merits further investigation.


Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Lymphoma, T-Cell, Cutaneous/drug therapy , Neoplasm Recurrence, Local/drug therapy , Trimetrexate/pharmacology , Aged , Aged, 80 and over , Antimetabolites, Antineoplastic/administration & dosage , Antimetabolites, Antineoplastic/adverse effects , Drug Resistance, Neoplasm , Female , Humans , Infusions, Intravenous , Lymphoma, T-Cell, Cutaneous/pathology , Male , Middle Aged , Treatment Outcome , Trimetrexate/administration & dosage , Trimetrexate/adverse effects
10.
Leuk Lymphoma ; 42(5): 969-79, 2001.
Article in English | MEDLINE | ID: mdl-11697652

ABSTRACT

The t(2;5)(p23;q35) or other rare chromosomal abnormalities involving 2p23 upregulate the ALK gene, which is not expressed in normal lymphocytes. Thus, detection of ALK protein is presumptive evidence of these 2p23 abnormalities. The t(2;5) and ALK immunoreactivity are common in anaplastic large cell lymphoma of T/null-cell lineage. However, a small subset of cases of Hodgkin's disease (HD) have been reported to either carry the t(2;5) or express ALK. In this study, we have immunohistochemically evaluated 327 cases of HD with the ALK-11 antibody. ALK-11 is a well characterized polyclonal antibody raised against an intracellular portion of the ALK protein. We detected ALK-11 immunoreactivity in 8 (2.4%) cases of HD. We further studied these positive cases with ALK-1 monoclonal antibody, which reacts with an intracellular portion of ALK, similar to ALK-11. All 8 ALK-11 positive cases were negative for ALK-1. These results indicate that rare cases of HD may react with ALK-11 antibody, similar to previous reports by others using different polyclonal anti-ALK antibodies. However, the absence of ALK-1 expression in these HD cases suggests that ALK protein is not truly present and that polyclonal anti-ALK antibodies may rarely yield non-specific cross reactivity. These results further support the use of anti-ALK antibodies in the differential diagnosis of HD from ALCL.


Subject(s)
Hodgkin Disease/diagnosis , Hodgkin Disease/enzymology , Protein-Tyrosine Kinases/analysis , Adolescent , Adult , Aged , Aged, 80 and over , Anaplastic Lymphoma Kinase , Antibodies , Child , Child, Preschool , Cross Reactions , Diagnosis, Differential , Disease-Free Survival , False Positive Reactions , Female , Humans , Immunohistochemistry , Lymphoma, Large-Cell, Anaplastic/diagnosis , Lymphoma, Large-Cell, Anaplastic/enzymology , Male , Protein-Tyrosine Kinases/immunology , Receptor Protein-Tyrosine Kinases , Tumor Cells, Cultured
11.
Am J Pathol ; 159(2): 527-35, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11485911

ABSTRACT

Anaplastic large-cell lymphoma (ALCL) of T- or null-cell lineage, as defined in the revised European-American lymphoma classification, includes a subset of tumors that carry the t(2;5)(p23;q35) resulting in overexpression of anaplastic lymphoma kinase (ALK). Patients with ALK+ ALCL are reported to have a better prognosis than patients with ALK- ALCL. Because the mechanisms for this survival difference are unknown, we investigated the hypothesis that apoptotic pathways may be involved. We therefore assessed expression levels of the anti-apoptotic proteins BCL-2 and BCL-XL and the pro-apoptotic proteins BAX and BCL-XS in T/null-cell ALCL using immunohistochemical methods and correlated the findings with ALK expression and apoptotic rate (AR), the latter assessed by a modified Tdt-mediated dUTP nick-end labeling assay. ALK was detected in 21 of 66 (31.8%) ALCLs. BCL-2 was not detected in 21 ALK+ ALCLs but was present in 26 of 45 (57.8%) ALK- ALCLs (P < 0.0001). ALK+ and ALK- ALCLs also showed significant differences in expression of BCL-XL, BAX, and BCL-XS. ALK+ tumors less commonly had a high level of BCL-XL (1 of 17 versus 14 of 35, P = 0.01), and more commonly had high levels of BAX (13 of 18 versus 15 of 36, P = 0.05), and BCL-XS (11 of 16 versus 12 of 31, P = 0.05) compared with ALK- tumors. ALK+ tumors also had a higher mean AR than ALK- tumors (3.4% versus 1.1%, P = 0.0002). Differential expression of BCL-2 family proteins may be responsible for the higher AR observed in ALK+ ALCL and provides a possible biological explanation for the better prognosis reported for patients with ALK+ ALCL.


Subject(s)
Genes, bcl-2 , Lymphoma, Large B-Cell, Diffuse/genetics , Lymphoma, Large B-Cell, Diffuse/pathology , Protein-Tyrosine Kinases/genetics , Proto-Oncogene Proteins c-bcl-2/genetics , Adolescent , Adult , Aged , Anaplastic Lymphoma Kinase , Apoptosis , Cell Division , Child , Child, Preschool , Chromosome Mapping , Chromosomes, Human, Pair 2 , Chromosomes, Human, Pair 5 , Female , Humans , Immunohistochemistry , In Situ Nick-End Labeling , Male , Middle Aged , Mitotic Index , Neoplasm Staging , Prognosis , Protein-Tyrosine Kinases/analysis , Proto-Oncogene Proteins/analysis , Proto-Oncogene Proteins c-bcl-2/analysis , Receptor Protein-Tyrosine Kinases , Translocation, Genetic , bcl-2-Associated X Protein , bcl-X Protein
12.
Anticancer Res ; 21(2A): 991-9, 2001.
Article in English | MEDLINE | ID: mdl-11396193

ABSTRACT

BACKGROUND: Breast cancer is characterized by complex genetic alterations found in multiple chromosomal regions, most commonly losses of 17p, 16q, 8p and others. A number of tumor suppressor genes mapped on these loci have been investigated in mammary tumors, whereas other gene products are of unclear function and await identification. MATERIALS AND METHODS: We analyzed the loss of heterozygosity (LOH) of two chromosomal loci: a. 16q24.3 using the genetic markers D16S303, D16S3026 and D16S3407 and b. 16q22.1, the locus of E-cadherin gene, using the microsatelite markers D16S503, D16S752 and D16S512, in a series of 63 sporadic invasive breast carcinomas consisting of 56 ductal, 4 lobular and 3 tumors of mixed type. Our findings were correlated with proliferative activity, ploidy and hormonal status of the tumors. RESULTS: Fourteen (22.2%) tumors demonstrated LOH of 16q24.3. Allelic imbalance of the 16q22.1 locus was found in 19 of 61 informative cases (31%) and commonly coexisted with LOH of 16q24.3. A significant association was observed between LOH of D16S752 and the absence of progesterone receptors in tumor cells (p = 0.005). CONCLUSIONS: LOH of 16q24.3 and 16q22.1 are frequent genetic alterations in breast cancer and they do not seem to correlate with tumor cell proliferation or ploidy. The statistical association between LOH of 16q24.3 and progesterone receptors need to be further investigated in larger series.


Subject(s)
Breast Neoplasms/genetics , Carcinoma, Ductal, Breast/genetics , Carcinoma, Lobular/genetics , Chromosomes, Human, Pair 16 , Loss of Heterozygosity , Adult , Aged , Aged, 80 and over , Alleles , Allelic Imbalance , Breast Neoplasms/metabolism , Carcinoma, Ductal, Breast/metabolism , Carcinoma, Lobular/metabolism , DNA, Neoplasm/analysis , Female , Humans , Microsatellite Repeats , Middle Aged , Neoplasm Invasiveness , Ploidies , Receptors, Progesterone/metabolism
13.
Haematologica ; 86(3): 274-81, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11255274

ABSTRACT

BACKGROUND AND OBJECTIVES: Interleukin-10 (IL-10) is a pleiotropic cytokine which increases bcl-2 levels and protects cells from steroid or doxorubicin-induced apoptosis. Hodgkin and Reed-Sternberg (HRS) cells bear functional IL-10 receptors. Thus serum IL-10 (sIL-10) might inhibit apoptosis in HRS cells, which could occur as a result of either chemotherapy or the crippled immunoglobulin genes. DESIGN AND METHODS: We determined sIL-10 levels in 122 patients with Hodgkin's lymphoma (HL), treated with ABVD or equivalent regimens with or without radiotherapy, and correlated them with presenting clinical and laboratory features, as well as failure-free survival (FFS) and overall survival. RESULTS: Elevated sIL-10 levels ( > or = 10 pg/mL) were detected in 55 patients (45%), and were correlated with advanced stage and elevated serum b2-microglobulin levels. At 7 years FFS was 85% vs. 63% for patients with normal vs. elevated sIL-10 levels, respectively (p=0.01); overall survival was 97% vs. 73% (p=0.005). Multivariate analysis with Cox's proportional hazards model demonstrated that elevated sIL-10 levels were the strongest independent predictor of FFS, and were also associated with inferior overall survival. INTERPRETATION AND CONCLUSIONS: We conclude that sIL-10 levels are elevated in 45% of patients with HL, and are associated with inferior FFS and overall survival, independently of other established prognostic factors.


Subject(s)
Hodgkin Disease/diagnosis , Interleukin-10/blood , Adolescent , Adult , Aged , Aged, 80 and over , Biomarkers/blood , Female , Hodgkin Disease/blood , Humans , Male , Middle Aged , Prognosis , Survival Rate
14.
Mod Pathol ; 12(11): 1062-71, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10574604

ABSTRACT

In order to understand better the possible role of cell-cycle regulating molecules in the pathogenesis of Hodgkin's disease (HD), the immunohistochemical distribution pattern of p16INK4A was investigated and compared with pRb, p53, and MDM2 protein status in 44 HD cases. Our findings were correlated to the presence of Epstein-Barr virus as detected by RNA in situ hybridization and clinicopathological parameters. p16INK4A protein immunoreactivity was found in all 44 cases with a proportion of Hodgkin-Reed-Sternberg (HRS) cells ranging from 30 to 90%. In 93% of the cases studied, pRb was detected in HRS, whereas all cases showed overexpression of p53. Almost all specimens (98%) were MDM2-positive as evaluated by 1B10 and/or IF2 monoclonal antibodies. EBER 1/2-transcripts were detected in 31.8% (14 of 44) of the examined samples. A significant correlation was observed between immunoreactivity of p16INK4A and MDM2 and the number of HRS cells (P = .0012 and P = .018, respectively). In a subgroup of cases, with p16INK4A expression in more than 50% of HRS cells, the percentage of pRb-positive neoplastic cells was inversely related to that of p16-positive ones (P = .007). No clinicopathological parameters or clinical prognostic indicators, including duration of response to therapy, were statistically related to the expression levels of any of the four proteins investigated or the presence of Epstein-Barr virus. Our findings suggest that p16 and pRb are regularly expressed and that their pathway in cell-cycle machinery seems to be intact in HD. However, further investigation is needed to shed light on the involvement of cell-cycle molecules in Hodgkin's lymphomagenesis and longer patient follow-up is required for conclusive prognostic correlation.


Subject(s)
Gene Expression Regulation, Neoplastic/physiology , Genes, p16 , Herpesvirus 4, Human/isolation & purification , Hodgkin Disease/genetics , Nuclear Proteins , Adolescent , Adult , Aged , Female , Herpesvirus 4, Human/genetics , Hodgkin Disease/virology , Humans , Immunohistochemistry , In Situ Hybridization , Male , Middle Aged , Neoplasm Proteins/genetics , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-mdm2 , RNA, Viral/genetics , Retinoblastoma Protein/genetics , Treatment Outcome , Tumor Suppressor Protein p53/genetics
15.
Cancer ; 87(4): 216-23, 1999 Aug 25.
Article in English | MEDLINE | ID: mdl-10455210

ABSTRACT

BACKGROUND: Cytologic distinction between follicle center lymphoma (FCL) and mantle cell lymphoma (MCL) is difficult with cytomorphology alone and requires immunophenotyping. The current study describes the distinction between follicle center and mantle cell lymphoma made with fine-needle aspiration (FNA) material. METHODS: One hundred ten cases primarily diagnosed and classified on FNA material as centroblastic-centrocytic (CBCC) and centrocytic (CC) non-Hodgkin lymphomas (NHLs) (Kiel classification) were included in the study. An additional retrospective immunocytochemical analysis was performed on frozen cytospin preparations using the monoclonal antibodies Bcl-2, CD10, CD5, CD23, CD43, and immunoglobulin M. RESULTS: The initial diagnostic workup classified 106 cases as CBCC-NHL and 4 as CC-NHL. The immunophenotype Bcl-2(+), CD10(+/-), CD5(-), CD23(-/+), CD43(-) was observed in 93 of 106 previously reported CBCC NHLs. In 11 of 106 cytospin preparations, neoplastic B cells expressed the CD5 pan T marker and, as a group, showed the pattern Bcl (+/-), CD10(-/+), CD5(+), CD23(-), CD43(+), which is considered typical of MCL. Based on the additional immunocytochemical data, all but 2 of the tumors were reclassified as FCL (n = 93) and MCL (n = 15). The mean proliferation fraction measured by MIB-1 (Ki-67) immunoreactivity was 16.3% and 17.5% in FCL and MCL, respectively. The revised cytopathologic diagnosis correlated significantly (P < 10(-9)) with the histology of 65 patients who underwent surgical excision biopsy. CONCLUSIONS: Subclassification of follicle-derived low grade NHL can be established with high accuracy on FNA material if cytomorphology is corroborated by a complete immunophenotypic analysis, which can be performed on both fresh and frozen stored cytospin material. The currently used criteria can be applied to aspirated cells for a conclusive cytopathologic diagnosis of MCL, which is of great clinical importance. Cancer (Cancer Cytopathol)


Subject(s)
Biopsy, Needle , Lymphoma, Follicular/diagnosis , Lymphoma, Non-Hodgkin/diagnosis , Adult , Aged , Aged, 80 and over , Antibodies, Monoclonal , Biomarkers, Tumor/analysis , Cell Count , Female , Humans , Immunoenzyme Techniques , Immunophenotyping , Ki-67 Antigen/analysis , Lymph Node Excision , Lymph Nodes/chemistry , Lymph Nodes/pathology , Lymphoma, Follicular/chemistry , Lymphoma, Follicular/classification , Lymphoma, Non-Hodgkin/chemistry , Lymphoma, Non-Hodgkin/classification , Male , Middle Aged
17.
Br J Cancer ; 77(3): 374-84, 1998.
Article in English | MEDLINE | ID: mdl-9472631

ABSTRACT

The present study represents a continuation of previous works in which we observed that lung carcinomas co-expressing MDM2 protein and p53 mutants (mt p53) exhibited more aggressive behaviour. In the above studies, we suggested a 'gain of function' mechanism of mt p53 proteins based on the fact that the MDM2 gene possesses a p53-responsive element (MDM2-p53RE). In this study, to prove our hypothesis, we selected 12 cases from a series of 51 bronchogenic carcinomas. In these 12 cases, we examined the ability of the expressed mt p53 to bind the MDM2-p53RE and correlated the findings with MDM2 expression. Furthermore, we constructed four of these p53 mutants and studied their transactivation properties by co-transfecting them with a reporter plasmid carrying MDM2-p53RE in the p53 null non-small-cell lung carcinoma cell line (NSCLC) H1299. We observed mutant p53 protein DNA-binding activity, which depended on the nature and the position of the amino acid substitution. The fact that the cases with DNA-binding activity were accompanied with MDM2 protein isoforms' overexpression is indicative of a 'gain of function' phenotype. This hypothesis was enforced by the findings of the transfection experiments, which revealed that certain p53 mutants enhanced the expression of the luciferase reporter gene either directly or indirectly via a dominant positive effect on the wild-type p53. In conclusion, this work is one first attempt to examine if the deregulation of the p53/MDM2 autoregulatory feedback loop is due to novel properties of certain p53 mutants in the specific environment of a subset of bronchogenic carcinomas.


Subject(s)
Lung Neoplasms/genetics , Nuclear Proteins , Proto-Oncogene Proteins/genetics , Tumor Suppressor Protein p53/physiology , DNA/metabolism , Humans , Immunohistochemistry , Mutation , Proto-Oncogene Proteins/analysis , Proto-Oncogene Proteins/physiology , Proto-Oncogene Proteins c-mdm2 , RNA, Messenger/analysis , Transfection , Tumor Suppressor Protein p53/analysis
18.
J Pathol ; 180(2): 129-37, 1996 Oct.
Article in English | MEDLINE | ID: mdl-8976869

ABSTRACT

Forty-one bronchogenic carcinomas were investigated for expression of MDM2 protein isoforms and their relationship to p53 protein levels and p53 gene alterations using molecular and immunohistochemical techniques. The findings were correlated with the pathological features of the carcinomas. MDM2 protein was overexpressed in 26 cases (63 percent). Western blot analysis with two monoclonal antibodies, 1B10 and IF2, revealed three MDM2 protein isoforms, p90, p57 and p76/74. p90 and p57 are capable of interacting with p53 protein, while p76/74 is not. Various patterns of MDM2 isoforms were seen. Although no correlation between the patterns and pathological features was observed, lymph node metastases were more frequent in the cases with MDM2 overexpression (P < 0.005). In 3 out of 17 specimens of normal lung tissue examined, there was a low level of expression of p90. Molecular analysis revealed that MDM2 overexpression was a consequence of increased transcription rather than MDM2 gene amplification. p53 protein was overexpressed in 21 cases (51 percent) and p53 gene alterations (mutations + allelic deletions) were detected in 23 patients (56 percent). A high degree of concordance (76 percent) between p53 mutations and p53 staining was noticed (P < 10(-5)). p53 gene alterations were significantly associated with lymph node disease (P < 0.01). MDM2 and p53 proteins were simultaneously detected in 21 cases (51 percent), of which 17 (42 percent) showed p53 and MDM2 overexpression. The latter group was positively correlated with p53 mutations (P < 0.05). A strong correlation between MDM2/p53 co-expression and lymph node metastases was observed (P < 0.001). The findings suggest that MDM2 overexpression is a common event in bronchogenic carcinoma. The selective expression of some MDM2 isoforms in neoplastic tissue and not in the surrounding normal areas underscores the pathological role of the various MDM2 products. Finally, the coexistence of MDM2 protein(s) and p53 aberrations (mutations and/or overexpression) in a subset of lung carcinomas may be indicative of a 'gain of function' phenotype, with more aggressive characteristics.


Subject(s)
Carcinoma, Bronchogenic/metabolism , Lung Neoplasms/metabolism , Nuclear Proteins , Proto-Oncogene Proteins/metabolism , Tumor Suppressor Protein p53/metabolism , Blotting, Northern , Blotting, Western , Carcinoma, Bronchogenic/pathology , Genes, p53/genetics , Humans , Immunohistochemistry , Lung Neoplasms/pathology , Lymphatic Metastasis , Mutation , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins c-mdm2 , Tumor Suppressor Protein p53/genetics
19.
Mod Pathol ; 9(5): 544-54, 1996 May.
Article in English | MEDLINE | ID: mdl-8733770

ABSTRACT

In this study, we investigated immunohistochemically the expression of mdm-2 protein in 93 surgically resected bronchogenic carcinomas. The findings were correlated with p53 protein detection status and clinicopathologic data (histologic type, differentiation grade of the lesions, lymph node metastases, and smoking history of the patients). Thirty of the 93 immunohistochemically examined specimens were subjected to Northern blot and differential polymerase chain reaction analysis to look into the mechanism of mdm-2 overexpression. Finally, we studied the concordance between p53 immunohistochemical positivity and p53 gene alterations as assessed by the single-strand conformation polymorphism technique. Seventy-three (78%) and 67 (72%) of 93 carcinomas showed nuclear immunoreactivity for mdm-2 and p53 proteins, respectively. We observed a high degree of concordance (75%) between p53 mutations and p53 immunolabelling, which was even higher in the specimens with p53 positively in more than 50% of the cells (90%). Despite the high percentage of mdm-2 and p53 expression, the two molecules were simultaneously detected in 50 (54%) of 93 cases. Forty-two (84%) of the 50 cases were accompanied by p53 mobility shifts, which indicated mutations. Interestingly, statistical analysis revealed an almost significant correlation between the carcinomas with mdm-2/p53 coexpression and lymph node disease (P = 0.058), which indicated a possible "gain of function" phenotype. In addition, absence of reactivity for both proteins was statistically more frequent in the patients without lymph node disease (P = 0.006). The mdm-2-positive/p53-negative immunohistochemical profile was more often seen in adenocarcinomas (P = 0.003), especially in well-differentiated ones (P = 0.02), than in other histologic types of lung cancer, which suggested a p53-independent pathway of mdm-2 overexpression. Molecular analysis showed that mdm-2 overexpression was a consequence of increased transcription rather than of mdm-2 gene amplification. The smoking history of the patients was strongly related to p53 (P = 10(-4)) even in the group of adenocarcinomas (P = 0.012). No correlation was observed between cigarette consumption and mdm-2 immunoreactivity.


Subject(s)
Carcinoma, Bronchogenic/chemistry , Carcinoma, Bronchogenic/genetics , Lung Neoplasms/chemistry , Lung Neoplasms/genetics , Nuclear Proteins , Proto-Oncogene Proteins/genetics , Adenocarcinoma/chemistry , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Aged , Blotting, Northern , Carcinoma, Bronchogenic/pathology , Carcinoma, Small Cell/chemistry , Carcinoma, Small Cell/genetics , Carcinoma, Small Cell/pathology , Carcinoma, Squamous Cell/chemistry , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , Female , Humans , Immunohistochemistry , Lung Neoplasms/pathology , Lymphatic Metastasis/immunology , Male , Middle Aged , Neoplasm Proteins/genetics , Polymerase Chain Reaction , Polymorphism, Single-Stranded Conformational , Proto-Oncogene Proteins c-mdm2 , Tumor Suppressor Protein p53/biosynthesis
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