Clara Sambade

Survivin is required for stable checkpoint activation in taxol-treated HeLa cells

Survivin is an essential chromosomal passenger protein whose function remains unclear. Here, we have used RNA interference to specifically repress Survivin in cultured HeLa cells. Immunoblot analysis showed that Survivin was no longer detectable in cultures 60 hours after transfection with Survivin-specific siRNA. Live cell analysis showed that many Survivin-depleted cells were delayed in mitosis, and immunofluorescence analysis of fixed specimens revealed that Survivin-depleted cells accumulated in prometaphase with misaligned chromosomes. The chromosomal passenger proteins, INCENP and Aurora-B, which can interact directly with Survivin, were absent from the centromeres of Survivin-depleted cells. These data contribute to the emerging picture that Survivin operates together with INCENP and Aurora-B to perform its mitotic duties. Some Survivin-depleted cells eventually exited mitosis without completing cytokinesis. This resulted in a gradual increase in the percentage of multinucleated cells in the culture. Time-lapse imaging of synchronized cultures revealed that control and Survivin-depleted cells arrested in mitosis in the presence of nocodazole; however, the latter failed to arrest in mitosis when treated with taxol. Immunofluorescence studies revealed that Survivin-depleted cells were unable to stably maintain BubR1 at the kinetochores in the presence of either taxol or nocodazole. Our data reveal that Survivin is not required for the spindle assembly checkpoint when it is activated by the loss of microtubules. However, Survivin is required for the maintenance of the checkpoint when it is activated by taxol, which is generally thought to cause a loss of spindle tension.

Solitary fibrous tumour of the thyroid: clinicopathological, immunohistochemical and ultrastructural study of three cases.

We describe three cases of solitary fibrous tumour (SFT) arising from thyroid stroma. Grossly, the tumours were clearly delimited but only partly encapsulated. The following histomorphological growth patterns were observed: bundles of cells in storiform configuration; non-structured bundles; prevalence of fibrous matrix; highly cellular, non-structured; prevalence of loose, non-structured extracellular substance; cellular proliferation and vascular spaces in a haemangiopericytic configuration and a lipomatous component. Immunohistochemical investigation demonstrated intense, diffuse vimentin positivity and focal, less intense actin positivity in all three cases. At electron microscopy we observed a primitive cell of mesenchymal type, with cytoplasm poor in organelles and rich in filaments; this cell sometimes presented differentiation characteristics. SFT is at present the most correct term for the lesions presented here despite some morphological characteristics which differ from cases reported in the literature.

Poorly Differentiated Carcinomas of the Thyroid Gland A Review of the Clinicopathologic Features of a Series of 28 Cases of a Heterogeneous, Clinically Aggressive Group of Thyroid Tumors

We evaluated the clinicopathologic, immunohistochemical, lectin histochemical, ultrastructural and morphometric characteristics of a series of 28 poorly differentiated carcinomas of the thyroid (PDCT). The 28 tumors were classified according to their predominant growth pattern as insular (n=12), trabecular (n=10) and solid subtypes (n=6). The overall mortality rate was 46%. No significant differences were found among the 3 subtypes. Data obtained by lectin histochemistry, electron microscopy, and morphometry point to a closer relationship of PDCT to follicular than to papillary carcinoma. Our results do not support the subdivision of PDCT into subtypes.

Hyalinizing trabecular adenoma: A misnomer for a peculiar tumor of the thyroid gland

We describe the clinical, histological, immunohistochemical, and electron microscopical features of 9 tumors fulfilling the criteria of the so-called hyalinizing trabecular adenoma (HTA) of the thyroid. Six tumors had the characteristic histology of HTA throughout, whereas in the remaining 3 tumors the classic pattern was identified focally in otherwise typical or atypical follicular adenomas. In one case, there was a focus of tumoral tissue outside the capsule, and in another there was a regional lymph node metastasis. Seven tumors were immunoreactive for thyroglobulin and cytokeratins, 1 tumor was positive for thyroglobulin and negative for cytokeratins, and another was negative for thyroglobulin and positive for cytokeratins. Scattered cells immunoreactive for neurotensin and somatostatin were found in 2 cases. Every tumor stained for S100 protein and neuron-specific enolase, but none showed immunoreactivity for calcitonin, calcitonin gene-related peptide, or chromogranin. The irregularity of the nuclear contours, the prominence of the cytoplasmic bundles of intermediate filaments, and the accumulation of basal lamina material around the neoplastic cells without the interposition of a well-defined basal lamina were the most distinctive electron microscopical features. The cytogenetic study performed in one case revealed, apart from cells with a normal karyotype, two abnormal clones: one with a translocation of chromosomes 2-3 and another with the same translocation and trisomies of chromosomes 7 and 12. Our results show that most HTAs display follicular cell differentiation and very low clinical aggressiveness. They also show that some HTAs are able to coexpress follicular cell and neuroendocrine markers and may behave like malignant neoplasms. We conclude that hyalinizing trabecular tumor is a more appropriate generic term than HTA to designate this relatively heterogenous group of lesions of the thyroid gland.

Limited synergistic effect of antisense oligonucleotides against bcr-abl and transferrin receptor mRNA in leukemic cells in culture

The synergistic use of antisense oligonucleotides (ASOs) towards the bcr-abl and the transferrin receptor (TfR) mRNA was studied in a chronic myeloid leukemia (CML) cell line, aiming to improve the efficiency of individual ASO treatment. At 20 microM concentration, bcr-abl ASOs reduced cell growth by 40% and was specific for cells that have the translocation: there was a 34% reduction of BCR-ABL protein. The TfR ASO reduced cell growth by 20% and decreased TfR protein by 24%. The ASOs were more potent at reducing cell growth when used in combination (respectively, -20 and -17% than bcr-abl ASO and TfR ASO when used individually at the 10 microM concentration), thus we postulate that there is synergism of action. Cell cycle analysis also revealed that the sub-G1 peak was bigger in the synergistic treatment.

The effects on growth and survival of IL-6 can be dissociated in the U-266-1970/U-266-1984 and HL407E/HL407L human multiple myeloma cell lines.

Several studies have documented IL‐6‐dependent growth promotion of murine and human neoplastic plasma cells. However, it is well known that human multiple myeloma (MM) cells in vitro show a considerable degree of heterogeneity concerning growth and survival requirements. This heterogeneity, which probably reflects overlapping effects of feeder cells, interleukin 6 (IL‐6) and components of fetal calf serum (FCS) as well as tumour heterogeneity in vivo, has hampered the elucidation of molecular mechanisms underlying the effects of IL‐6. In an attempt to dissociate growth and survival promotion of IL‐6, we have studied two pairs of human MM cell lines, HL407E/HL407L and U‐266‐1970/U‐266‐1984, selected to represent different stages of in vitro tumour progression and dependence of feeder cells and exogenous IL‐6. We demonstrated that exogenous IL‐6, in the presence of FCS, conveyed: (a) a strong growth stimulatory effect with weak or no survival promotion in HL407L and U‐266‐1970 cells; (b) promotion of survival with no effects on growth in HL407E cells; (c) no growth or survival promotion to U‐266‐1984. Moreover, our results suggested that IL‐6 may enhance apoptosis in U‐266‐1970/U‐266‐1984 cells, and that FCS may interfere with IL‐6 in its growth stimulatory effect. The relative dissociation of growth, survival and apoptotic effects of IL‐6 leads to the conclusion that the HL407E/HL407L and U‐266‐1970/U‐266‐1984 pairs of cell lines provide a useful human model system to study molecular mechanisms underlying these separate events.

Follicular and papillary variants of medullary carcinoma of the thyroid.

Two medullary carcinomas of the thyroid (MCT) with relatively unusual patterns are reported. The first was an aggressive tumour which occurred in a 66-year-old man and displayed in most areas follicular structures. The second tumour occurred in a 36-year-old woman, followed a benign course and showed papillary infoldings lined by multilayered neoplastic cells. The search for thyroglobulin yielded negative results whereas calcitonin immunoreactivity could be found in most neoplastic cells of both tumours. The diagnosis of MCT was further substantiated by the presence of scarce amyloid deposits and typical neuro-secretory granules by electron microscopy. These cases demonstrate once more that follicular and papillary structures can be a prominent feature of some MCTs reinforcing therefore the major role of immunocytochemistry in the differential diagnosis of thyroid carcinomas. Papillary MCT seems to carry a good prognosis in contrast to follicular MCT if one takes into account the follow-up data of the present cases together with those of similar cases reported in the literature; the whole series is nevertheless too small to allow for definite conclusions on this matter.

U-2940, a human B-cell line derived from a diffuse large cell lymphoma sequential to Hodgkin lymphoma

Several patterns of association between Hodgkin and non‐Hodgkin lymphomas are recognized, some of which support a common cellular origin or shared transformation events for both malignancies. We describe the U‐2940 cell line derived from a diffuse large B‐cell lymphoma with some features consistent with mediastinal large B‐cell lymphoma, clinically apparent 1 month after the initial course of chemotherapy for Hodgkins disease, fulfilling the criteria for composite malignancies. U‐2940 cells display a mature B phenotype with hypermutated IgH rearrangement typical of germinal/postgerminal center origin. The cell line is negative for Epstein‐Barr virus and no evidence of t(14;18) was found. U‐2940 cells display multiple chromosomal rearrangements similar to recurrent aberrations described in both Hodgkin and non‐Hodgkin lymphomas, also partially shared by U‐2932 derived from a B‐cell lymphoma sequential to Hodgkins disease. The original large B‐cell lymphoma and the U‐2940 cell line bear microsatellite instability, an abnormality associated with particular subtypes of non‐Hodgkin lymphomas and found in tissues involved by Hodgkin lymphoma. Therefore, U‐2940 cells bear several features known to occur in Hodgkin and in non‐Hodgkin lymphomas, leading to the assumption that this cell line may constitute a useful tool to address elective pathways of lymphomagenesis and eventually the Hodgkin and non‐Hodgkin lymphoma association.

Reed-Sternberg cells form by abscission failure in the presence of functional Aurora B kinase.

Large multinucleated Reed-Sternberg cells (RS) and large mononucleated Hodgkin cells (H) are traditionally considered to be the neoplastic population in classical Hodgkin lymphoma, (cHL) and postulated to promote the disease. However, the contribution of these larger cells to the progression of cHL remains debatable. We used established cHL cell lines and cHL cellular fractions composed of small mononucleated cells only or enriched in large RS/H cells to investigate RS/H cell origin and to characterize the cells which they derive from. We confirm that the small mononucleated cells give rise to RS/H cells, and we show that the latter proliferate significantly more slowly than the small cells. By using live-cell imaging, we demonstrate that binucleated RS cells are generated by failure of abscission when a few small cells attempt to divide. Finally, our results reveal that the small mononucleated cells are chromosomally unstable, but this is unlikely to be related to a malfunctioning chromosomal passenger protein complex. We propose that the small mononucleated cells, rather than the RS/H cells, are the main drivers of cHL.

Molecular pathology in the diagnosis of hematologic neoplasia

Over the past decade molecular genetic methods have played an increasingly important role in the diagnosis of hematologic malignancies. Moreover, they have provided a tool to analyze many of the non‐random cytogenetic anomalies associated with hematologic neoplasias, contributing considerally to our understanding of several of those diseases, and to improving diagnostic accuracy. The rapid development of molecular genetics progressively allows the replacement of time‐consuming and technically demanding procedures. Even more relevant are the new clinical applications that already include the search for valuable prognostic information and ways of evaluating minimal residual disease or recognizing early relapsing disease. This paper is a critical but necessarily simplified overview of the main contributions of molecular genetics to the field of hematopathology. We discuss the information provided by several molecular methods within different clinical contexts, covering common problems in diagnostic pathology as well as prognostic evaluation and therapy monitoring.