Aleksandra Grzanka

Cytoskeletal reorganization during process of apoptosis induced by cytostatic drugs in K-562 and HL-60 leukemia cell lines

The aim of the present study was to investigate the reorganization of F-actin, vimentin and tubulin in K-562 and HL-60 cell lines during apoptosis induced by etoposide, doxorubicin and taxol. The distribution of cytoskeletal proteins was analyzed by fluorescence microscopy. Actin was also studied by confocal microscopy and at the ultrastructural level. Changes in the distribution of cytoskeletal proteins were found to be dose-dependent and appeared to be more intense in HL-60 cells. Etoposide- and doxorubicin-treated cells showed similar changes in the distribution of F-actin, vimentin and tubulin. The reorganization of cytoskeletal proteins seemed to be consistent with features of apoptosis. An increase in bright staining of F-actin, vimentin and tubulin at the site of apoptotic bodies formation was observed. Immunogold labeling of actin in HL-60 cells was associated with features typical for apoptosis, i.e. compaction and margination of nuclear chromatin. K-562 cells showed cytoplasmic actin-positivity in the cytoplasm. Significant changes in morphology of HL-60 cells were found in the following concentrations: etoposide 20, 200 microM; doxorubicin 5, 10 microM and taxol 2-10 microM. The investigated proteins seemed to be involved in the above-reported apoptotic changes. Bright staining of F-actin, vimentin and tubulin, concentrated at the site of apoptotic bodies formation might suggested importance of these proteins for this process. Moreover, the increase in actin labeling in areas of chromatin compaction and margination of nuclear chromatin especially in HL-60 cells, which are more susceptible to apoptosis might implicate that actin might be involved in the chromatin remodeling during apoptosis.

Expression of cyclin A, B1 and D1 after induction of cell cycle arrest in the Jurkat cell line exposed to doxorubicin.

Jurkat human lymphoblastoid cells were incubated in increasing concentrations of doxorubicin (0.05, 0.1 and 0.15 μM) to induce cell death, and their expression of cyclin A, B1 and D1 was evaluated by flow cytometry (cell cycle progression, Annexin V assay, percentages and levels of each of the cyclins), transmission electron microscopy (ultrastructure) and confocal fluorescence microscopy (expression and intracellular localization of cyclins). After low‐dose doxorubicin treatment, Jurkat cells responded mainly by G2/M arrest, which was related to increased cyclin B1, A and D1 levels, a low level of apoptosis and/or mitotic catastrophe. The influence of doxorubicin on levels and/or localization of selected cyclins was confirmed, which may in turn contribute to the G2/M arrest induced by the drug.

Doxorubicin-induced F-actin reorganization in cofilin-1 (nonmuscle) down-regulated CHO AA8 cells

The actin cytoskeleton plays an important role in many cellular processes, including cell mortality, mitosis, cytokinesis, intracellular transport, endocytosis and secretion but also is involved in gene transcription. The dynamics of the actin cytoskeleton is controlled by different classes of actin-binding proteins (ABPs) which regulate the polymerization of actin filaments. In this report we used siRNA against cofilin-1 (nonmuscle) to demonstrate the effect of cofilin on the nuclear and cytoplasmic actin pools in CHO AA8 cells after exposition to various concentrations of doxorubicin. The immunofluorescence studies showed doxorubicin dose dependent tendency to formation the multinucleated giant cells, but also the increase of fluorescence intensity of cofilin in nuclei of untransfected cells. Induction of cell death with doxorubicin treatment in untransfected cells revealed both mitotic catastrophe (in both lower and higher doxorubicin doses) and apoptosis (mostly in higher doxorubicin doses), whereas among cofilin-1 down-regulated cells we observed only mitotic catastrophe. The results suggest that cofilin has apoptosis-inducing ability and that mitotic catastrophe is independent from F-actin content in cell nucleus. In this point of view we conclude that different mechanisms of chromatin reorganization are involved in these two processes. Moreover, we suppose that apoptosis and mitotic catastrophe are independent from each other.

Merkel cell carcinoma: an illustrative case and review

Merkel cell carcinoma (MCC) was first described by Toker in 1972, as trabecular carcinoma [1, 2]. It is a primary cutaneous tumor of neuroendocrine origin characterized by aggressive course and poor prognosis [3–5]. Agelli and Clegg in 2007 showed that the incidence of MCC in the U.S. was 0.24/100000 per year [6]. Merkel cell carcinoma has a high propensity for local recurrence, lymphatic spread and distal metastases. Metastases are usually found in the skin (28%), liver (13%), bones (10%), and brain (6%). Typically, at the time of diagnosis, local or distant metastases are present. Merkel cell carcinoma affects mainly the elderly, more often men, usually between 65 and 85 years of age. Primary lesions are frequently localized in sun-exposed areas. In 29–40% of cases it is the head and neck region, followed by extremities (21–38%), trunk (7–23%), and other skin regions (3.4–12%) [7]. Merkel cell carcinoma often arises in the setting of immunodeficiency (post-transplant immunosuppression or HIV infection), autoimmune connective tissue diseases and neoplasm, particularly Hodgkins disease, B-cell lymphoma, chronic lymphocytic leukemia, breast and ovary cancer [8, 9]. Established risk factors for MCC development are UV radiation, immunosuppression and Merkel cell polyomavirus infection [7, 10]. Clinically MCC appears as an indolent, rapidly growing blue-red nodule often with telangiectasias. Histological findings are: monomorphous indistinct bluish cells, often arranged in trabeculae or strands with numerous mitotic figures, apoptotic cells and occasionally necrosis. Lymphocyte intra- and peritumoral infiltration is common. Routine histological examination may be of limited diagnostic value. Immunohistochemical staining, particularly against cytokeratin 20 (CK20) or chromogranin A, increase the effectiveness of MCC diagnosis [11]. Therapeutic management of choice is wide surgical excision or Mohs micrographic surgery of the tumor with sentinel lymph node biopsy. Adjuvant radiotherapy or chemotherapy is administered according to the clinical staging of disease. Metastases are treated with protocols similar to small-cell lung carcinoma management [12, 13]. A 74-year-old woman presented to our clinic with blue-red colored, well-demarcated skin tumors ranging from 0.5 cm to 2.0 cm in diameter located on the left lower extremity. Lesions were hard and painful on palpation (Figure 1). The enlarged inguinal lymph nodes were present bilaterally. Additionally the patient had a history of arterial hypertension, type 2 diabetes, rheumatoid arthritis and post-thrombotic syndrome. Figure 1 Blue-red colored, hard and painful to the touch skin tumors of the left lower leg diagnosed as MCC Lesions appeared 2 years ago, initially they would remit spontaneously. One year after the first occurrence, a nodular, ulcerated lesion located in the proximity of the left medial malleolus was biopsied. Histopathological examination of skin biopsy revealed positive staining for chromogranin A and CD56 as well as positive staining for cytokeratin 7 and cytokeratin 20 with a dot-like pattern. Deep surgical margin was positive. During current hospitalization skin biopsy was repeated revealing nests of small undifferentiated cells with round nuclei and scant cytoplasm. Numerous mitotic figures and apoptotic cells were present with occasional necrosis. Abundant peritumoral lymphocyte infiltration was observed. Immunohistochemical stainings were positive for CK20 (with a characteristic dot-like pattern), CD56, epithelial membrane antigen (EMA, MUC1), neuron-specific enolase (NSE, focal expression). Leukocyte common antigen (LCA) expression was positive only in peritumoral infiltrate (Figure 2). Adjacent muscular tissue was infiltrated with tumor cells. Based on clinical appearance and histology, MCC was diagnosed. Figure 2 Merkel cell carcinoma. A, B – Hematoxylin and eosin staining. The obtained result of the histopathological examination found within the dermis area is low-differentiated small cancer cells with scanty cytoplasm and round nuclei with small grains. ... Routine laboratory blood and urine tests, X-ray and computed tomography (CT) scans of the thorax, chest examination, USG of the abdomen and histology of enlarged inguinal lymph nodes were normal. The patient was staged IIC T4 N0 M0, where IIC is for primary tumors > 2 cm in size with extracutaneous invasion, T4 stands for primary tumor invading the bone, muscle, fascia, or cartilage; N0 – no regional lymph node metastasis and M0 – no distant metastases. The patient has undergone two surgeries with skin grafting. Due to local spread of the tumor, the 2nd and 1st fingers with metatarsal head were amputated. Currently adjuvant chemotherapy is considered. Merkel cell carcinoma is a rare neuroendocrine skin tumor occurring in the elderly, more often in men (70%). Common localization is the head and neck area and limbs, several cases of MCC in the anogenital area and on the mucosae have been reported [14]. Clinical appearance of MCC is heterogeneous. It frequently presents as an asymptomatic, reddish, bluish, or purple tumor of the skin. The size at the time of the first consultation is usually smaller than 2 cm, although rapid growth is characteristic [15, 16]. Merkel cell carcinoma pathogenesis remains largely unknown, but ultraviolet radiation and immunosuppression may play a significant role in the development of this cancer. In recent years, the relationship between Merkel cell polyomavirus infection and the development of the tumor was observed [17]. In the patient presented in this report, the incidence of tumors on both legs and the history of spontaneously resolving nodules may indicate MCC metastases without an apparent primary tumor. Spontaneous regression of the primary MCC tumor is not uncommon, with a dozen of cases described in medical literature [18]. Enlarged inguinal lymph nodes in our patient could indicate changes in tumor spread via lymphatic vessels. Cases of micro-metastases in the lymph nodes without clinical lymphadenopathy have been reported as well. Therefore, the sentinel lymph node biopsy and chest and abdomen imaging are necessary. Ulceration is uncommon in MCC. We believe that coexistence of MCC with post-thrombotic syndrome in our patient may explain ulceration of MCC tumor in this case. Merkel cell carcinoma derives from neuroendocrine cells and typically has appearance of ‘blue-cell tumor’ comprised of small, monomorphous cells with scant cytoplasm. Cancer cells are usually restricted to the dermis and subcutaneous tissue with a little propensity to invade epidermis. Differential diagnosis should consider basal cell carcinoma, squamous cell carcinoma, lymphoma, melanoma, metastatic neuroblastoma and neuroendocrine carcinoma. Useful diagnostic features are a positive dot-like pattern of staining for CK20 and sometimes other cytokeratins as well as positive staining for chromogranin A, somatostatin, gastrin characteristic of cells of neuroendocrine origin. Merkel cell carcinoma cells also exhibit a positive reaction with CD117, CD99, but negative with LCA and S-100 protein and of TTF-1. In our case, MCC was positive for cytokeratin 7, CK20 chromogranin A and MUC1. The prognosis in MCC is usually poor. The size of the primary tumor below 2.0 cm is associated with better prognosis, unfortunately, because of the very rapid proliferation of tumor cells, and diagnostic difficulties delaying diagnosis, in most cases, patients are diagnosed with MCC at the stage when the primary lesion exceeds 2.0 cm [19]. The classification of TMN American Joint Committee on Cancer (AJCC) proposed a clinical staging of MCC (0 to IV) [20]. According to this classification, the estimated 5-year survival rate for patients with stage IIC T4 N0 M0 is 50%. Merkel cell carcinoma lesions are considered highly malignant, hence a combination of surgery, radiotherapy in stages IA to IIIB of the disease is recommended [21–24]. Because of a rapid progression of the disease, adjuvant chemotherapy is frequently administered [2]. One can consider both the chemotherapy and radiotherapy in order to reduce the tumor mass prior to surgery in stages IIC to IIIB. In our patient, due to the presence of coexisting diseases and general condition, only surgical treatment was applied. In the IV stage of disease, the treatment of choice is palliative chemotherapy with the assessment of response to therapy and toilet surgery or radiotherapy of the bone, central nervous system and extensive skin metastases. Because of its similarity to small lung cancer, recommended chemotherapy protocols are cisplatin with etoposide or doxorubicin and cyclophosphamide or ifosfamide. The value of adjuvant radiotherapy has been confirmed with meta-analysis [25–27].

Polish Lymphoma Research Group Experience With Bexarotene in the Treatment of Cutaneous T-Cell Lymphoma

Bexarotene, a synthetic retinoid licensed for the treatment of refractory cutaneous T-cell lymphoma (CTCL), has been used clinically in Poland since 2007 in 21 patients. The objective of our retrospective, multicenter study was to evaluate our experience with bexarotene therapy, including efficacy, safety, and survival outcomes. We retrospectively identified 21 adult patients who were treated with bexarotene between the years 2007 and 2012. Starting dose of bexarotene was 300 mg/m2 per day. The analysis included 3 patients with early-stage mycosis fungoides (MF), 16 patients with advanced-stage MF, and 2 patients with Sézary syndrome (SS). The mean duration of therapy with bexarotene was 14.5 months. Use of bexarotene resulted in an overall response rate of 81.0%, although the overall mortality rate was 52.8%. In our study, early-stage CTCL responded better than advanced-stage CTCL (100.0% vs. 77.8%, respectively). The mean time to observable response was 1.8 months, and the mean duration of the response was 16.4 months. Most significant side effects were hyperlipidemia, hypothyroidism, and a bleeding gastric ulcer. Based on the results of our analysis, bexarotene is a valuable tool in the treatment of refractory early-stage CTCL. Although a majority of patients initially responded to therapy, the high mortality rate in the advanced-stage group suggests that bexarotene does not completely resolve the therapeutic problems in all stages of CTCL. Patient stratification for bexarotene treatment may need a thorough reassessment, in that bexarotene may not be an effective drug in the very advanced stages of CTCL.

Folliculitis decalvans and orofacial granulomatosis

Folliculitis decalvans (FD) is a rare and chronic inflammatory dermatosis which pathogenesis remains unclear. It is classified as primary neutrophilic cicatricial alopecia presented clinically with follicular pustules and papules, hemorrhagic crusts, erosions and scarring within scalp. Orofacial granulomatosis (OFG) is a recurrent or persistent orofacial swelling histopathologically presented with non-caseating granulomas. The etiology of the disease is not fully understood. There are hypotheses concerning pathogenesis of the disease including genetic, allergic, infectious and immunological factors. We report a case of a patient with FD and concomitant orofacial granulomatosis successfully treated with dapsone. A 32-year-old man was admitted to the Department of Dermatology to diagnose follicular pustules, erosions, yellow scales, crusts with scarred areas and focal hair loss within the scalp, especially within vertex and accompanying persistent lip swelling with normal appearance of the tongue and no oral lesions (Figure 1). Moreover, we observed erythrosquamous lesions and papules on the lateral surfaces of the trunk, within groins and armpits. The lesions on the scalp occurred in childhood and the lip swelling persisted for 3 years. Except skin eruptions the patient did not report any other complaints. He was initially treated in another clinic for seborrheic dermatitis and acne inversa topically with antibiotics, glucocorticosteroids, tacrolimus and orally with antibiotics (doxycycline), antihistamines with temporary improvement. The patient suffered from post-traumatic epilepsy and was treated with carbamazepine. He had a craniotomy due to head injury (concussions and subdural hematoma of the left temporo-parietal brain region, basilar skull fracture and squamous part of the right temporal bone fracture) during the traffic accident 20 years earlier. During the hospitalization routine laboratory tests showed an elevated C-reactive protein level and erythrocyturia which occurred to be irrelevant (the patient was consulted by the urologist). Swabs from erosions and pustules were collected and two types of bacteria were cultured – Staphylococcus aureus and Streptococcus agalactiae. To exclude concomitant systemic diseases, we performed additional examinations. There were no abnormalities in abdominal ultrasonography or chest X-ray. Antinuclear antibodies, lupus anticoagulant, cardiolipin antibodies (IgG and IgM), b2-glycoprotein 1 antibodies (IgG and IgM), anti-Borrelia spp. antibodies tests (IgG and IgM), concentration of C1 esterase inhibitor and Quantiferon-TB Gold test in patient’s serum were negative or within normal

Pyoderma gangrenosum mimicking inflammatory breast cancer

Address for correspondence: Agnieszka Białecka MD, Chair of Dermatology, Sexually Transmitted Diseases and Immunodermatology, Faculty of Medicine, Nicolaus Copernicus University, 9 Skłodowskiej-Curie St, 85-094 Bydgoszcz, Poland, phone: +48 696 557 558, +48 535 854 568, fax: +48 52 585 40 18, e-mail: agnieszka_bialecka@wp.pl Received: 2.08.2016, accepted: 5.09.2016. Pyoderma gangrenosum mimicking inflammatory breast cancer

Follow-up of patients with mycosis fungoides after interferon α2b treatment failure.

Introduction Treatment of T cell cutaneous lymphoma( CTCL) is a controversial subject and the effectiveness of treatment is still low. Aim Report of single center experience of management CTCL after progression after first line treatment. Material and methods We present 41 patients with CTCL, 29 received interferon α2b in first line, and 12 of them received second line therapy. Results Overall response rate for second line therapy was 60%. Conclusions Results of the follow-up of patients with mycosis fungoides after interferon α2b treatment failure with the literature review and discussion.