Flow cytometric DNA content analysis of neoplastic cells in haemolymph of the cockle Cerastoderma edule

Epizootiological outbreaks of disseminated neoplasia (DN) have been reported in association with mass mortalities in various bivalve species including the cockle Cerastoderma edule. A flow cytometric (FCM) procedure to study DNA content was successfully adapted and tested in haemolymph cells (haemocytes and neoplastic cells) of the cockle. The FCM results were similar to those obtained by histological analysis (DN diagnosis and haemolymph cell features). FCM analysis revealed differences in DNA content among normal haemocytes (diploid) and neoplastic cells. Four types of cells with abnormal DNA content were found in the haemolymph of affected animals: hypodiploid, hyperdiploid, triploid-sesploid and pentaploid. Our results suggest that the flow cytometric DNA content analysis can be applied to identify neoplastic cell types and to study the association between different cell types and the DN progression or remission in this edible and commercially important bivalve species.     

Centrosome-centriole abnormalities are markers for abnormal cell divisions and cancer in the transgenic adenocarcinoma mouse prostate (TRAMP) model

We utilized the transgenic adenocarcinoma mouse prostate (TRAMP) model to study the formation of abnormal mitosis in malignant tumors of the prostate. The results presented here are focused on centrosome and centriole abnormalities and the implications for abnormal cell divisions, genomic instability, and apoptosis. Centrosomes are microtubule organizing organelles which assemble bipolar spindles in normal cells but can organize mono-, tri-, and multipolar mitoses in tumor cells, as shown here with histology and electron microscopy in TRAMP neoplastic tissue. These abnormalities will cause unequal distribution of chromosomes and can initiate imbalanced cell cycles in which checkpoints for cell cycle control are lost. Neoplastic tissue of the TRAMP model is also characterized by numerous apoptotic cells. This may be the result of multipolar mitoses related to aberrant centrosome formations. Our results also reveal that centrosomes at the poles in mitotic cancer cells contain more than the regular perpendicular pair of centrioles which indicates abnormal distribution of centrioles during separation to the mitotic poles. Abnormalities in the centriole-centrosome complex are also seen during interphase where the complex is either closely associated with the nucleus or loosely dispersed in the cytoplasm. An increase in centriole numbers is observed during interphase, which may be the result of increased centriole duplication. Alternatively, these centrioles may be derived from basal bodies that have accumulated in the cell's cytoplasm, after the loss of cell borders. The supernumerary centrioles may participate in the formation of abnormal mitoses during cell division. These results demonstrate multiple abnormalities in the centrosome-centriole complex during prostate cancer that result in abnormal mitoses and may lead to increases in genomic instability and/or apoptosis.     

Death by apoptosis in salivary glands of females of the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae)

The salivary glands of females of the tick Rhipicephalus sanguineus at three feeding stages: unfed, engorged, and at day three post-engorgement, were subjected to cytochemical methods of enzymatic analysis and cell viability. Comparing glands at these stages, was observed distinct staining patterns in cells of different types of acini, specially in degenerating types III, II, I, which were affected in this sequence by cell death. This study also revealed changes in: nuclei, staining intensity for acid phosphatase and ATPase activities, and permeability of the plasma membrane. Acid phosphatase activity was inversely proportional to that of ATPase, while ATPase activity was always proportional to membrane integrity. The glands of unfed females exhibited high metabolic activity and cells with intact nucleus and plasma membrane, suggesting that the presence of acid phosphatase detected in these individuals may participate in the normal physiology of some acini, as they were not undergoing degeneration. In acini I and II of engorged females, we observed cells with intact membranes, as well as changes characterized by nuclear changes, decrease in ATPase activity, and stronger acid phosphatase activity. At day three post-engorgement, degeneration progressed to more advanced stages, loss of membrane integrity was observed in most cells (of some type I acini, most type II acini, and all type III acini), as well as prominent nuclear changes, decrease in ATPase activity, and intense acid phosphatase activity, resulting in apoptotic bodies. During the death of cells nuclear changes preceded cytoplasmic ones in the following sequence: nuclear changes, loss of ATPase activity, loss of integrity of the plasma membrane, increase in acid phosphatase activity, and formation of apoptotic bodies. The presence of acid phosphatase with a secondary role (late) during cell death, degrading final cell remnants, characterized this process in the glands of R. sanguineus females as atypical or non-classic apoptosis.     

A stochastic model of cancer initiation including a bystander effect

A stochastic model of cancer initiation is considered. The model is used to evaluate whether a bystander effect may be important in the pre-malignant and malignant stages of carcinogenesis, and furthermore, on the basis of epidemiological data, to estimate the mutation rates of genes involved in the development of oral leukoplakias. The bystander effect is defined here as the capability of oncogenic mutations to increase the mutation probability of neighbouring (bystander) cells, thus leading potentially to a cascade of neighbouring mutated and neoplastic cells as a pre-stage in the development to leukoplakias and cancer. We find that incidence data for oral cancer are indeed in accordance with a significant bystander effect, operating either alone or in combination with genomic instability in the early stages of carcinogenesis, i.e. the development of neoplasia. Simulations performed gave a picture of how mutations and neoplasia may spread in a tissue, to form characteristic leukoplakias with a core of neoplastic cells. The model also showed that the probability of finding at least one neoplastic cell in the tissue after a given number of years is more sensitive to changes in genomic instability within the cell itself than to changes in a bystander effect. Based on epidemiological data we also calculate the maximum number of oncogenic genes that may be involved in the bystander effect and development of genomic instability. Even if capable of explaining the initial development of oncogenic mutations towards neoplastic cells, the bystander model could not reproduce the observed incidence rates of leukoplakia without assuming a carcinogen mutation probability per cell per year of neoplastic cells practically equal to one. This means that the bystander effect, to be of substantial importance in the final development of neoplastic cells towards leukoplakias, requires a very significant increase in mutation probabilities for bystanders to neoplastic cells. Alternatively, additional mechanisms such as abnormal cell differentiation and uncontrolled proliferation and apoptotis in the neoplastic stage may be of major importance during the development to cancerization.     

Alternate pathogenesis of systemic neoplasia in the bivalve mollusc Mytilus.

The proliferative disease systemic neoplasia, also termed hemic neoplasia or disseminated sarcoma, was studied in four Puget Sound, Washington populations of the bay mussel (Mytilus sp.). Using flow cytometric measurement of DAPI-stained cells withdrawn from the hemolymph, DNA content frequency histograms were generated for 73 individuals affected by the disease. The cells manifesting systemic neoplasia were found to exist as either of two separate types, characterized by G0G1 phase nuclear DNA contents of either approximately 4.9 x haploid (pentaploid form) or approximately 3.8 x haploid (tetraploid form). The two disease forms were found to coexist in all four mussel populations sampled, with overall relative prevalences of 66% pentaploid form, 29% tetraploid form, and 5% exhibiting both disease forms simultaneously. These findings represent the first unequivocal demonstration of multiple cell types in a bivalve neoplasia. The two forms appear to represent separate pathogenetic processes rather than sequential stages of a single pathogenesis. Two cell cycling parameters associated with proliferative activity were employed to compare the alternate forms: (i) the percentage of cells assigned to the DNA Synthesis (S) phase of the neoplastic cell cycle, and (ii) the proportion of neoplastic cell mitotic figures in hemocytological preparations. Mean values for both parameters were significantly higher for mussels with the tetraploid form of the disease, suggesting a higher rate of proliferation relative to the pentaploid form. Qualitatively, cells of the tetraploid form contained slightly lower nuclear and cytoplasmic volumes compared to those of the pentaploid form. An observed wide variation in neoplastic cell nuclear size within either disease form may reflect the distribution of cells in the G0G1, S, and G2M phases of the cell cycle. Potential etiologic relationships between the two forms are discussed.     

Cytochemistry of nucleoproteids and some cathionic proteins in the peripheral blood leukocytes of patients with lung cancer.

In 40 patients with untreated lung cancer cytochemical studies of the peripheral blood leukocytes were conducted by means of a cytological method for the simultaneous staining of nucleoproteids (RNP and DNP) and some cathionic proteins (after Zvetkova and Zvetkov [60]). Changes were detected in the RNP cytoplasmic contents of lymphocytes, of which the most outstanding were the reduction and uneven distribution of RNP granules, their frequent extracellular expulsion by means of microclasmatoses, as well as changes in the staining of cathionic proteins of RNP accompanied by an increased nuclear chromatin condensation in the small and medium-sized lymphocytes. Parellel to reducing of the percentage of these cells in the peripheral blood of patients with advanced neoplastic disease an increased number of lymphoblastoid and monoblastoid cells is established with RNP diffusely stained, but reduced in quantity and localized in the cytoplasmic periphery and projections (compared to Downey type II atypical cells). By means of one of the variants of the method (modified type of Feulgen's reaction) a characteristic distribution and structuring of the nuclear chromatin is established in mono- and polymorphonuclear cells, most clearly expressed in the nuclei of monocytes and monoblastoid cells, as well as in nuclei of neutrophil granulocytes. In these cellular types a more specific nuclear modelling (microhypersegmentation) is observed resulting in multiple irregular nuclear projections on the nuclear surface, probably caused by subkaryolemal distribution of uneven chromatin thickenings. The changes are also recorded in the cathionic protein containing secondary cytoplasmic granules in granulocytes-neutrophils and eosinophils, probably associated with changes in the lysosomal and phagocytic functions of these cells in neoplastic diseases. The authors discuss the importance of the obtained results in connection with data on the participation of lymphocytes and neutrophils in the immune response to tumour antigenic stimuli during the course of the neoplastic process, as well as with data on the suppressive effect of antigenic (serum, viral) factors, possibly affecting the synthesis and the transport of cellular nucleoproteids (RNP and DNP) in leukocytes of cancer patients.     

Multidimensional slit-scan detection of bladder cancer. Preliminary clinical results

A multidimensional slit-scan flow system was developed for the automated recognition of abnormal cells derived from cancer of the uterine cervix and its precursors. It provides great sensitivity in both its ability to recognize cellular abnormality and to deal with the myriad potential causes of false alarms in an automated flow system. While its initial application was the automated recognition of the spectrum of neoplasia in gynecologic cytology samples, a preliminary study was carried out using specimens obtained from the urinary bladder. Cellular material was collected by bladder irrigation and stained with the fluorochrome acridine orange. One hundred fifty-three bladder irrigation specimens, including 115 abnormal specimens containing cells derived from neoplastic lesions of the bladder epithelium, were analyzed. For the purposes of this study, abnormal specimens from the urinary bladder included specimens containing cells derived from the following lesions of the urothelium: dysplasia (atypical hyperplasia), carcinoma-in-situ, and transitional cell carcinoma, grades 1-3. Approximately 50,000 cells were analyzed for most specimens. Of the 38 presumed normal specimens (specimens containing only normal urothelial components), four were instrument classified abnormal. For the 69 specimens containing cells derived from transitional cell carcinoma, grade 1, 1-2, 2, 66 were correctly classified as abnormal while three were classified as normal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of neuron-specific (gamma gamma) enolase in proliferating (supportive and neoplastic) Schwann cells. An immunohisto- and electron-immunocyto-chemical study of ganglioneuroblastoma and schwannomas

Neuron-specific (gamma gamma) enolase, a glycolytic enzyme used as a relatively specific marker for normal neurons and neuroendocrine cells, has recently been found in a variety of neoplastic cells and in reactive astrocytes. Its localization was investigated by immunohisto- and electron-immunocyto-chemistry, in the proliferating supportive Schwann cells of a peripheral ganglioneuroblastoma and in the neoplastic Schwann cells of four acoustic tumours. By light microscopy, the neoplastic Schwann cells showed moderate uneven diffuse immunopositivity for enolase. By electron-immunocytochemistry, both types of Schwann cells demonstrated immunopositivity discretely limited to their cell surface membranes. The neoplastic ganglion cells and axons of the ganglioneuroblastoma and the normal neurons and axons included in the schwannomas were, as expected, intensely immunopositive. The visualization of gamma gamma enolase on the cell surface membranes of both neoplastic and non-neoplastic proliferating Schwann cells suggests that increased glycolytic activity may occur on the surface of these proliferating cells irrespective of the nature of the proliferation.     

Malignant melanoma of the cervix. Report of a case

A malignant melanoma of the cervix, a rare neoplasm, was found to have an unusual cytologic pattern, similar to that of a leiomyosarcoma. A biopsy sample was diagnosed as cervical malignant melanoma of the spindle cell type. Some neoplastic cells in the tissue contained melanin pigment, whereas none of the abnormal cells in the cervical scrapes, except for an abnormal giant cell, had visible cytoplasmic pigment. The abnormal cells in the cervical scrape specimen were spindle-shaped, as were their nuclei, which is why the cytologic pattern was interpreted as that of a leiomyosarcoma. Evidence from this and previously reported cases shows that malignant melanoma must be considered as a possible source of exfoliated abnormal nonpigmented spindle-shaped cells in a cervicovaginal cell sample.     

Chromatophoromas in a pine snake

Iridophoroma and melanophoroma were diagnosed in an adult male pine snake. Light microscopic examination of irregularly thickened white and black portions of abnormal scales demonstrated two distinctive populations of pigment-containing cells. Pigment cells within abnormal-appearing white scales had needle-shaped granules that were dark amber in color while black portions were composed of pigment cells typical of melanophores, with dark black, round granules. Both populations of cells showed junctional activity, and clusters of both neoplastic pigment cell types were found in adjoining areas of the epidermis. By electron microscopy, the pigment cell with amber-colored granules contained reflecting platelet profiles typical of iridophores while pigment cells with dark round granules contained melanosomes. At a junctional area between abnormal white and black scales, mosaic chromatophores containing reflecting platelet profiles and melanosomes were observed. At 1 1/2 years following initial diagnosis, the snake died and neoplastic iridophores were found at multiple visceral sites; there was no evidence of metastases of melanophores to any organ. The two pigment cell tumors are believed to have developed from either stem cells destined to become iridophores and melanophores or from prexisting iridophores and melanophores in the dermis.     

Microtubule organization and cell division in embryogenie protoplast cultures of white spruce (Picea glauca)

Summary Immunofluorescence methods were developed for examining the distribution of microtubules in freshly isolated and cultured protoplasts and regenerated somatic embryos of white spruce (Picea glauca). Freshly isolated protoplasts consisted of both uniand multinucleate types. Uninucleate protoplasts established parallel cortical microtubules during cell wall formation and cell shaping, divided within 24 h and developed into somatic embryos in culture. Dividing cells were characterized by preprophase bands (PPBs) of microtubules, atypical spindle microtubules focused at the poles and a typical phragmoplast at telophase. Multinucleate protoplasts also established parallel arrays of cortical microtubules during cell wall formation. In addition their nuclei divided synchronously within 4 days, then cell walls formed between the daughter nuclei. Individual multinucleate protoplast-derived colonies subsequently gave rise to elongate suspensor cells thereby forming embryo-like structures by 7 days.     

Argyrophilic nucleolar organizer regions (AgNORs) in interphases and metaphases of normal and neoplastic gill cells of Macoma balthica (Bivalvia: Tellinidae) from the Gulf of Gdansk, Baltic Sea

Chromosome analysis of gill cells of different populations of Macoma balthica (L.) from the Bay of Gdansk (Baltic Sea) revealed 2 clam categories, 1 with neoplastic features and 1 without. Silver-staining was performed on interphase and metaphase cells of both categories. The mean argyrophilic nucleolar organizer region (AgNOR) count per abnormal interphase cell was significantly higher than in normal interphase cells. Normal silver-stained metaphases had 3 nucleolar organizer region (NOR) chromosome phenotypes. The location of the NORs in the most frequent phenotype (55.6% in 54 metaphases scored) was interstitial on the largest metacentric chromosome pair, Pair No. 1. Abnormal silver-stained metaphases had a higher number of active NOR sites. Different phenotypes were observed (frequency greater than 10% for 67 metaphases scored); 2 were similar to those in normal metaphases and 5 were ectopic. The higher activity of AgNORs observed in abnormal cells confirmed the diagnosis of malignant neoplasia.     

Parasites and malignancies, a review, with emphasis on digestive cancer induced by Cryptosporidium parvum (Alveolata: Apicomplexa)

The International Agency for Research on Cancer (IARC) identifies ten infectious agents (viruses, bacteria, parasites) able to induce cancer disease in humans. Among parasites, a carcinogenic role is currently recognized to the digenetic trematodes Schistosoma haematobium, leading to bladder cancer, and to Clonorchis sinensis or Opisthorchis viverrini, which cause cholangiocarcinoma. Furthermore, several reports suspected the potential association of other parasitic infections (due to Protozoan or Metazoan parasites) with the development of neoplastic changes in the host tissues. The present work shortly reviewed available data on the involvement of parasites in neoplastic processes in humans or animals, and especially focused on the carcinogenic power of Cryptosporidium parvum infection. On the whole, infection seems to play a crucial role in the etiology of cancer.     

Ultrastructure of the uterus in an ovariectomized gecko (Hemidactylus turcicus) after administration of exogenous estradiol

The uterus of an oviparous gecko, Hemidactylus turcicus, was analysed after ovariectomized females underwent a period of treatment (up to 14 days) with exogenous estradiol. Analysis focused on the uterine mucosa, which is made up of an epithelial layer and an underlying lamina propria containing the shell glands. These tissues are thought to be responsible for secretion of the eggshell components and were thus chosen for analysis using transmission electron microscopy. In ovariectomized females, the epithelial layer was low and cuboidal with minimal/no differentiation or secretory activity. Treatment with exogenous estradiol resulted in a significant increase in cell height associated with gradual differentiation of the epithelium. Development of non-ciliated cells included production of secretory granules (low electron density) at the apical cell surface. The shell glands showed less obvious changes over the course of treatment. Shell glands contained two cell types: dark cells with darkly staining nuclei and organelles, and light cells with very indistinct nuclei and organelles, except for prominent rough endoplasmic reticulum and free ribosomes. This study provides results consistent with published light microscopy studies for other reptiles and additionally provides ultrastructural details of reptilian uterine development not previously available.     

CELL DIFFERENTIATION AND CARCINOGENESIS: A CRITICAL REVIEW*

Many resemblances exist between cell differentiation and neoplastic change: the importance of cytoplasmic factors is particularly evident, as the genotype appears to remain unmodified in the nuclei of differentiated as in malignant cells. There is no opposition between malignancy and differentiation, highly malignant cells retaining in many cases a differentiation very close to normal. Loss of differentiation in malignancy is only an epiphenomenon. While differentiation appears to be partially reversible, and in some models totally reversible, neoplastic changes are, with a very small number of exceptions, irreversible. Differentiation involves the gradual change of a group or a population of cells, malignancy of a single cell. Further work on the molecular basis of differentiation would help to understand the nature of neoplasia, and vice versa.     

Microtubule organization in lymphoid malignancies.

The abnormal organization of actin-containing microfilaments and vimentin-containing intermediate filaments in neoplastic lymphocytes of T and B cell origin has been described. We investigated microtubules of pathologic cells from 34 lymphoid malignancies, by immunofluorescence microscopy, using monoclonal tubulin antibody. In most cases, apart from two cases of lymphoma, one T cell lymphoma and one B cell lymphoma, interphase leukemia cells, lymphoma cells, and myeloma cells were shown to contain well-organized microtubules which were associated with a microtubule organization center at one end. In the cells of a patient with T cell lymphoma, although microtubules were not visible in the lymphoma cells from lymph nodes, they became visible after 72 hours in culture with concanavalin A (Con A) and interferon alpha. Cap formation was observed with antitubulin monoclonal antibody in the peripheral blood lymphocytes from a chronic lymphocytic leukemia patient, but well-developed microtubules were observed on other occasions in the same patient. There were no obvious structural differences between microtubules in T and B cell lymphoid malignancies, but leukemia cells and lymphoma cells with irregularly shaped nuclei, such as adult T cell leukemia cells and B cell lymphoma cells with cleaved nuclei, had complicated microtubules surrounding their irregular nuclei. In general, after blastogenic stimuli with phytohemagglutinin-P (PHA-P), Con A, and pokeweed mitogen (PWM), the development of the microtubules was proportional to the incorporation of 3H thymidine (3H-TDR). In most cases, after incubation with granulocyte colony-stimulating factor (G-CSF) and interferon alpha, the number of intact cells decreased and the number of degenerated cells increased, but the intact cells had intact microtubules.     

A case of germinoma in a limpet (Patella coerulea) (Patellogastropoda)

Germinoma is a gonadal neoplasm originating from progenitor cells in germinal epithelium. Frequently described in some populations of bivalve molluscs, to our knowledge, germinoma has never been reported in gastropods so far. In this paper we describe the histopathological findings of some atypical cellular masses, originating in the undifferentiated germ cell layer in the male gonads of a limpet (Patella coerulea), whose morphological appearance resembled that of a germ cell tumor. The abnormal, and independent growth with no evidence of maturation of the undifferentiated and atypical germ cells, the limited number of follicles involved (n < 10%) and the absence of tissue invasion, supported a diagnosis of Stage 1 germinoma.     

Effects of Carvacrol on a Human Non-Small Cell Lung Cancer (NSCLC) Cell Line, A549

Carvacrol, the predominant monoterpene in many essential oils of Labitae including Origanum, Satureja, Thymbra, Thymus, and Corydothymus has substantial antibacterial, antifungal, antihelmintic, insecticial, analgesic and antioxidant activities. Approximately 75% of lung cancer is non-small cell carcinoma (NSCLC) which comprises several histologic types squamous cell, adenocarcinoma and large cell carcinoma. It was reported that the portion of lung tumors diagnosed as denocarcinoma has increased. Thus a human non-small cell lung cancer (NSCLC) cell line, A549 was chosen for this study. To investigate the effects of carvacrol on cell morphology, apoptosis and total protein amount, the cells incubated with various concentration of carvacrol in DMSO for 24 h. In carvacrol applied A549 cell line, increase in dose of carvacrol caused a decrease in cell number, degeneration of cell morphology and a decrease in total protein amount. To characterize carvacrol induced changes in cell morphology, cells were examined by light microscopy. Cells were treated with carvacrol were seen to have detached from the disk, with cell rounding, cytoplasmic blebbing and irregularity in shape. The data demonstrate that carvacrol is very potent inhibitor of cell growth in A549 cell line.     

Changed expression of 9-O-acetyl GD3 (CDw60) in benign and atypical proliferative lesions and carcinomas of the human breast

 Expression of gangliosides is affected in various ways by malignant cell transformation. In the present study, we investigated the expression of CDw60, a constituent of O-acetylated disialogangliosides, in benign and atypical proliferative breast diseases, and preinvasive and invasive carcinomas by immunohistochemistry and thin-layer chromatography (TLC). In normal ducts, antibodies to CDw60 (mAb M-T21) reacted to membranes of the Golgi apparatus in the juxtaluminal cell compartment. A similar polarized distribution of Golgi cisterns in epithelial cells was observed in several benign lesions, i.e., fibroadenomas, intraductal papillomas, and gynecomastia. In contrast, blunt duct adenosis and duct hyperplasia exhibited an abnormal cytosolic and cell surface staining, whereas atypical duct hyperplasia showed randomly dispersed immunoreactive Golgi cisterns, indicating loss of epithelial polarity. In mammary carcinomas and in two breast carcinoma cell lines (MCF-7 and EFM-19) the neoplastic cells contained CDw60-immunolabelled Golgi complexes, which were distributed in a disorderly fashion throughout the cytoplasm, thus reflecting a loss of epithelial polarity. Additionally, only well differentiated ductal carcinomas in situ or invasive ductal carcinomas disclosed a strong cell surface labelling, which was absent in lower differentiated carcinomas of the same types. In all carcinomas, the intensity of CDw60 immunostaining decreased with progressing loss of differentiation (grade of dedifferentiation), as demonstrated by staining intensity in paraffin sections and by evaluation of the relative amounts of extracted 9-O-acetyl GD3 by TLC. Our results indicate that abnormal CDw60 expression is already detectable in benign proliferative breast lesions with different risk rates to develop into malignant lesions. Downregulation of CDw60 expression in poorly differentiated invasive carcinomas may be the consequence of loss of cell functions usually associated with poor prognosis. Received: 19 February 1998