Indian Journal of Dermatology
BASIC RESEARCH
Year
: 2011  |  Volume : 56  |  Issue : 5  |  Page : 501--504

Implications of Bax, Fas, and p53 in the pathogenesis of early-stage mycosis fungoides and alterations in expression following photochemotherapy


Fatma Aydin, Yildiz Levent, Senturk Nilgun, Yuksel Esra Pancar, Turanli Ahmet Yasar 
 Ondokuz Mayis University, School of Medicine, Departments of Dermatology and Pathology, Samsun, Turkey

Correspondence Address:
Fatma Aydin
Ondokuz Mayis University School of Medicine, Department of Dermatology, TR- 55139 Kurupelit, Samsun
Turkey

Abstract

Background: The underlying molecular basis of mycosis fungoides (MF) has not yet been clarified. However, defects in apoptosis may contribute to its pathogenesis. Aim: We investigated the expression of Bax, Fas, and p53 in early-stage MF patients and any alterations in expression following photochemotherapy. Materials and Methods : Bax, Fas, and p53 expressions were studied by immunohistochemistry in both keratinocytes and lymphocytes on paraffin-embedded skin specimens from 27 early-stage MF patients. Results: Bax, Fas, and p53 staining was shown in the lymphocytes in 0/27, 26/27, and 11/27 patients at the time of diagnosis, whereas these ratios were 0/27, 9/27, and 0/27, respectively, after psoralen plus ultraviolet A (PUVA) treatment. The decrease in p53 and Fas expression in the lymphocytes was found statistically significant. Bax, Fas, and p53 staining in the keratinocytes was shown in 5/27, 27/27, and 25/27 patients at the time of diagnosis, whereas these ratios were 0/27, 22/27, and 4/27, respectively, after PUVA treatment. The decrease in p53, Fas, and Bax expression in the keratinocytes was found statistically significant. Conclusion: Although Bax seems unrelated with early-stage MF, Fas and p53 expression in the lymphocytes may contribute to the understanding of the pathogenesis of this disease.



How to cite this article:
Aydin F, Levent Y, Nilgun S, Pancar YE, Yasar TA. Implications of Bax, Fas, and p53 in the pathogenesis of early-stage mycosis fungoides and alterations in expression following photochemotherapy.Indian J Dermatol 2011;56:501-504


How to cite this URL:
Aydin F, Levent Y, Nilgun S, Pancar YE, Yasar TA. Implications of Bax, Fas, and p53 in the pathogenesis of early-stage mycosis fungoides and alterations in expression following photochemotherapy. Indian J Dermatol [serial online] 2011 [cited 2019 Sep 19 ];56:501-504
Available from: http://www.e-ijd.org/text.asp?2011/56/5/501/87130


Full Text

 Introduction



Mycosis fungoides (MF) represents the commonest form of the cutaneous T-cell lymphoma (CTCL) subsets, which is a monoclonal neoplastic disorder of T-helper cell lineage. Several theories on the etiology of MF have been postulated, including exposure to environmental and infectious agents as well as genetic factors. The prevailing theory is that MF is likely to develop secondary to chronic antigenic stimulation due to a multitude of factors in a stepwise process involving mutations in oncogenes and certain DNA repair genes. [1],[2]

The override of apoptotic control is suspected to cause or contribute to some forms of carcinogenesis. In several skin tumors including MF, involvement of an apoptotic process in the proliferation or regression of the lesions has been shown. [3]

Bax protein is identified as a promoter of apoptosis. Cells displaying higher levels of Bax easily undergo apoptosis. [2] Fas is a transmembrane protein that mediates apoptosis. Cells with a mutated Fas gene are resistant to apoptosis and thus accumulate in lesional tissues. Defective Fas signaling has been suggested as a possible causative agent in MF pathogenesis due to defects in apoptosis signaling in skin-homing T-cells. [4] Abnormalities of cell cycle control genes and well-defined tumor suppressor genes such as p53 may also contribute to the disease pathogenesis, progression, and treatment resistance. p53 normally stimulates apoptosis, but when it is mutated or absent it favors cell survival. Abnormal expression of p53 protein in primary CTCL has been shown in the late stages of the disease. [5]

This study aimed at investigating by immunohistochemistry the expression of Bax, Fas proteins, and p53 in the pathogenesis of early-stage MF before and just after clinical remission was achieved with psoralen plus ultraviolet A (PUVA).

 Materials and Methods



Twenty-seven patients diagnosed clinically and histopathologically as MF (stages IA and IB) from May 1997 to November 2006 in the Dermatology Department of Ondokuz Mayis University Hospital, Samsun, Turkey, were included in this prospective study. Diagnosis of the disease was based on clinical-pathological criteria as generally accepted. Stage of disease was established according to TNM (tumor, node, metastases) classification of the National Cancer Institute Workshop on MF. [6]

This study was approved by the local ethical committee and informed consent was obtained from all patients enrolled in the study. None of the patients had a history of autoimmune disorder or other malignancies, and none progressed to the advanced stage of the disease during this short follow-up period. Physical examination and routine laboratory studies were performed every month. Routine examination included a complete blood cell count, antinuclear antibody titer, liver and renal function tests, erythrocyte sedimentation rate, and peripheral blood smear. An ophthalmologic examination was performed before initiation of PUVA therapy. Bone marrow aspiration and biopsy and computed tomography scans of thorax and abdomen were performed when indicated.

Light source and treatment regimen

All patients were treated with a standard PUVA regimen [Dermo Ringo (Kalfa Sun 100 W FR71 B/P B14S Rapid), MRD-Advantage, GmbH, Germany]. Patients ingested 8-methoxy-psoralen (8-MOP) 0.6 mg/kg 2 h before the UVA exposure. The initial UVA dose was administered according to skin phototype. Treatments were given three times a week until more than 90% clearing of the lesions had occurred. Once clinical remission was achieved, maintenance therapy was started and was gradually reduced according to clinical response. During the therapy and the follow-up period, only emollients were permitted for topical skin care. Cumulative doses and total number of treatments were noted.

Histological skin analysis

Bax, Fas, and p53 expressions were analyzed in both keratinocytes and lymphocytes in paraffin-embedded skin specimens from patients with early MF at the time of diagnosis and after clinical remission was achieved. Two cutaneous biopsies were obtained from each patient. Initial biopsies were taken before therapy from lesions located on sun-protected areas. The diagnosis of MF was confirmed histologically according to established criteria. The second biopsy specimens were obtained from areas in close proximity to the first lesions biopsied.

Routine histology

The specimens were fixed in 10% buffered neutral formalin solution for 24 h. Formalin-fixed biopsies were dehydrated in a graded alcohol series using an overnight tissue-processing schedule (Leica TP1020, Leica Microsystem). All of the tissues were embedded in paraffin and sectioned at a thickness of 4-6 μm (Leica 2125 rotary microtome, Leica Microsystem). The sections were stained with hematoxylin-eosin (HE) (Varistain 24-4, Shandon Inc.) and examined under light microscopy (Olympus BX51, Japan).

Immunohistochemical staining

For immunohistochemistry, tissue sections were deparaffinized and rehydrated. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in distilled water for 10 min. To retrieve the antigen epitopes, the sections were heated in microwave for 30 min in 0.01 mol/L citrate buffer solution (pH 6.0). The slides were then treated with protein block, serum-free (Dako, Carpinteria, CA, USA) for 5 min at room temperature. Slides were incubated with Bax (Neomarkers, CA, USA, clone 2D2), CD95 (Fas) (Neomarkers, CA, USA, clone 95C03) and p53 (Neomarkers, CA, USA, clone DO07) primary antibody for 30 min at room temperature. The sections were incubated with secondary antibody at room temperature for 30 min. The sections were developed with 3.3'-diaminobenzidine (DAB) and counterstained with Mayer's hematoxylin. Positive staining was semiquantitatively assessed. Staining of >10% of the cells (keratinocytes and lymphocytes) was accepted as positive.

Data management and statistical analysis

Statistical analyses were done using SPSS 13.0 for Windows. The descriptive statistics were indicated by mean ± SD and range values. Wilcoxon signed rank test was performed to compare changes in the expression of Bax, Fas, and p53 values before and after therapy. P- value of <0.05 was considered statistically significant. In addition, McNemar test was used to control the expressions in the second specimens that were previously regarded as positive.

 Results



Twenty-seven patients (16 men, 11 women) were included in this study. The mean age was 51.41 ± 11.6 years (range 26-70). All patients were in stage IA or IB according to the TNM classification for CTCL. There were no abnormalities in routine blood tests. No pulmonary or abdominal involvement was detected on computed tomographic scans of the thorax and abdomen in those patients in whom they were performed.

According to the classification of Fitzpatrick, five patients had skin type II, 18 patients had skin type III, and four patients had skin type IV. The mean number of PUVA treatments was 314.6 ± 155.4 (range 120-700). The total dose of PUVA was 774.8 ± 512.7 j/cm 2 (total±SD) (range 86-2025).

Although Bax expression remained unchanged in lymphocytes, p53 and Fas expression demonstrated a decrease after clinical remission had been achieved. This decrease in p53 and Fas expression in the lymphocytes was found statistically significant (P<0.05). In the keratinocytes, decreased expression of all these proteins was shown and this was also found statistically significant (P<0.05). [Table 1] gives the expression of Bax, Fas, and p53 in early stage MF patients before PUVA therapy and just after clinical remission was achieved.{Table 1}

 Discussion



Apoptosis is a form of cell death designed to eliminate unwanted host cells by a set of gene products and can be demonstrated in a wide variety of both benign and malignant skin tumors.

Early MF lesions develop from the accumulation of CLA + T-lymphocytes, and the initial event might be related to defective apoptosis occurring in a subset of activated T-cells expressing CLA. [3] It has been shown recently that UV treatment can induce apoptosis in several leukemic cell lines, and PUVA treatment is able to induce apoptosis in human T-lymphocytes, but the precise pathways and molecules involved remain largely unidentified. [7],[8]

p53 is a tumor suppressor gene that accumulates when DNA is damaged, and arrests the cell cycle to allow additional time for repair. Thus, p53 normally stimulates apoptosis, but when it is mutated or absent it favors cell survival. [9] As a result, tumor suppressor gene p53 may play a role in the pathogenesis of MF, which is a disease of lymphocyte accumulation. In the literature, p53 staining of lymphoid cells was reported infrequently in MF, and was largely limited to advanced cases. [10],[11] It has also been proposed as a prognostic factor because high p53 protein expression is seen in high grade but not in low-grade CTCL. [12] Dereure et al. showed nuclear staining of p53 in the lymphocytes in two of 12 MF cases; they also demonstrated increased p53 expression in the nucleus of basal keratinocytes above the neoplastic infiltrate in some MF cases. [3] Li et al. reported increased p53 protein accumulation in advanced stages of CTCL regardless of p53 gene mutations and proposed that p53 may play a role in disease progression with mechanisms other than gene mutations, in most cases. [11] Beylot-Barry et al. studied 61 skin biopsy specimens of 54 patients with primary cutaneous lymphoproliferative disorders. They reported no expression of p53 in 14 MF and seven pleomorphic T-cell lymphomas of either low- or high-grade malignancy. They found no p53 overexpression in the epidermotropic T-cell lymphomas. [13]

In our study, 25 and 11 of 27 patients showed p53 staining in the keratinocytes and lymphocytes, respectively. After PUVA treatment, p53 staining was not detected in any of the patients. Different ratios of p53 staining were obtained in different studies and ours could be explained by the homogeneous disease stage distribution among patients. Since loss of p53 expression in the lymphocytes at the time of remission was found statistically significant, we propose that p53 may play a role in the pathogenesis of MF. In view of the percentage of the patients expressing p53 in the early stage of MF, we also suggest that p53 might be more evident in advanced cases of the disease. This significant change in p53 staining after treatment in both keratinocytes and lymphocytes may strongly point to the effect of PUVA through apoptosis.

Fas is the main signaling membrane molecule involved in postactivation T-lymphocyte apoptosis. [14] A membrane-bound or soluble ligand, called Fas ligand, produced by cells of the immune system, binds to Fas on the T-cells and activates a death program. [9] Dereure et al. found a significantly lower level of Fas expression on peripheral blood CD4 lymphocytes in patients with CTCL compared with subjects with benign inflammatory cutaneous disorders and with healthy donors. [14] Zoi-Toli et al. also demonstrated decreased Fas expression in aggressive types of CTCL. [15] These data suggest that loss of Fas receptor expression may be involved in the pathogenesis or progression of some types of CTCL. However, in contrast, Kamarashev et al. showed that the malignant cells of the dermal infiltrate express Fas and Fas ligand, sometimes at a high level, in MF and Sezary syndrome. [16]

In this study, we also found high level of Fas expression. Fas was detected in lymphocytes of 26/27 early stage MF patients and only nine patients showed Fas staining after PUVA treatment; the decrease was found to be statistically significant. The increased expression of Fas and significant decrease in staining after treatment lead us to think that Fas is important in MF pathogenesis. Another explanation for high Fas expression at the time of diagnosis is viral infections, which have been shown to lead to increased Fas and/or Fas L-expression and increased sensitivity to Fas/Fas L-dependent apoptosis. Increased Fas expression may support the role of viruses in the etiology of MF. The decrease in Fas expression in keratinocytes after PUVA treatment could be a result of the effect of PUVA on apoptosis.

The other agonist of apoptosis is Bax. By selective binding to bcl-2, it alters its activities and promotes apoptosis. [9] The study of Bax by Dereure et al. does not favor a role of alterations in this gene in MF pathogenesis. [3] In our study, no Bax expression was determined in the lymphocytes, whereas only five patients showed staining in the keratinocytes. This finding is in accordance with previous findings and may be due to earlier stages of the disease.

There are a few studies about apoptosis in MF patients and these usually include patients with different disease stages. However, disease stage was homogeneous in our group of patients. We suggest that expression of Fas in the lymphocytes might be implicated in the pathogenesis of early stage MF. Although expression of p53 is not strongly related with early stage MF, different pathogenetic routes may function in MF, involving p53. Bax seems unrelated with early stage MF. This study is unique in that it investigates apoptotic markers at the time of the diagnosis and just after clinical remission was achieved. Our major outcome was demonstration of a significant decrease in Fas expression in the lymphocytes after PUVA treatment, so this finding may be used in treatment follow-up of MF patient who are in clinical remission. Future studies may focus in this area. Further studies with larger number of cases are needed to better elucidate the role of these oncogenes in the pathogenesis of MF.

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