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Malignant tumors are known to have an heterogeneous cell population. Metastases are supposed to be formed by subpopulations with high metastatic capability. These metastatic cells constitute the so called invasive phenotype. In that order of ideas, metastases could be pathologically different from their parent tumors if the invasive phenotype had distinct morphological features. The similarities or differences between primary tumors and their metastases have not been adequately studied at the ultrastructural level. In this work we report an electron microscopic study of liver leiomyosarcoma metastases which shows alterations not described in primary tumors of that kind.

Biopsies of liver metastases from a colon leiomyosarcoma were surgically obtained. Samples were processed with routine techniques for transmission electron microscopy and observed in a Hitachi H-500 electron microscope.

Some alterations usually observed in primary leiomyosarcomas were seen. They were presence of myofilaments with focal densities, nuclear changes, swollen mitochondria, and abundance of rough endoplasmic reticulum (Fig. 1).

Scientific Program

C-ret proto-oncogene encodes a protein in the tyrosine kinase (TK) family of transmembrane receptors for growth factors. C-ret expression has been identified in normal tissues and tumors of neural crest origin such as inherited MEN (multiple endocrine neoplasia) syndromes, particularly medullary thyroid cancers where there are confirmed germ-line mutations. Gene rearrangement has been observed in papillary thyroid carcinomas (PTC) and has been shown to have cytoplasmic localization of the altered gene product. C-ret proto-oncogene product is also necessary for the normal development of the peripheral nervous system, enteric nervous system and the kidney. A recent profile of the TK receptors in prostate cancer (PCA) identified c-ret protein in prostate cancer tissue and xenograft derived from human PCA. This study is the first to report the immunohistochemical (IH) evaluation of c-ret in human prostatic tissue.

Thirty radical prostatectomy specimens for PCA were formalin-fixed, sectioned at 5mm and paraffin-embedded.

Microscopy FATAL ERROR -- Missing abstract.amp; Microanalysis Sponsoring SocietiesFATAL ERROR -- Missing abstract.mdash;August 1997

Vitamins C (VC) and K3 (VK3) and a VC: VK3 combination with a VC: VK3 ratio of 100:1 were assayed for their antitumor activity against a DU 145 human prostate cancer cell line. Co-administration of the vitamins enhanced the antitumor activity 20-25 fold even with a lh exposure time. While exogenous catalase destroyed the antitumor activity, hydrogen peroxide-induced lipid peroxidation was negligible. The results of additional assays revealed: a transient increase in ATP production, a decrease in DNA synthesis, and increase in protein synthesis and a decrease in thiol levels. Subsequently the DU145 cells were treated with each vitamin alone or with the vitamin combination at their 50% cytotoxic doses and then examined using transmission electron microscopy. Treatment of DU145 cells with the vitamin combination exacerbated the cytotoxic effects induced by individual vitamin treatment.

Untreated DU145 cells show numerous mitochondria whose pale matrices are in sharp contrast to the cytoplasmic background (Fig. 1).

Microscopy FATAL ERROR -- Missing abstract.amp; Microanalysis Vendor DirectoryFATAL ERROR -- Missing abstract.mdash;August 1997

Laser-induced fluorescence (LIF) spectroscopy of tissues has been proposed as an adjunct to endoscopy for in vivo gastrointestinal (GI) diagnosis of premalignant lesions. In previous studies, under ultraviolet excitation (350-370 nm), sources of LIF signal differences in normal, and premalignant colonie tissues have been identified including 1) changes in gross tissue morphology, 2) increased hemoglobin absorption in adenomatous tissues, and 3) increased red fluorescence in dysplastic cells. However, many questions remain regarding the specific origins and biochemical correlates of GI tissue autofluorescence.

We report on a study of quantitative ultraviolet LIF spectral microscopy in human colonic tissues using a laser scanning confocal microscope with argon laser excitation 351-364 nm. Frozen sections (6μm) of normal colon (n=10) and tubular adenoma (n=8) were prepared from fresh surgical resection specimens using a previously published protocol. To identify histological components accurately, LSCAM slides were alcohol fixed and H&E histologically stained following confocal imaging.

Identification of altered gene expression in colonie preneoplastic lesions may play a key role in early detection and prevention of colon cancer. Considering carcinogen treated rat colonie mucosa harbors a mixed population of both normal and aberrant crypts, it is of interest to independently examine the genetic profile of these two populations. The present study was designed to use a novel method whereby RT-PCR was performed on alcohol fixed ACF which were micro dissected from normal surrounding crypts.

Male F344 rats were injected with azoxymethane, a colon specific carcinogen, at a dose of 15 mg/kg body weight once weekly for 3 weeks. After 28 weeks colons were excised, flushed with RNAse free ice cold phosphate-buffered saline, fixed flat on filter paper, fixed in 70% ethanol and immediately frozen at -80°C After a minimum 24 hours colons were placed on the lid of a sterile, clear petri dish filled with ice.

Microscopy FATAL ERROR -- Missing abstract.amp; Microanalysis Vendor DirectoryFATAL ERROR -- Missing abstract.mdash;August 1997

Author Index

There have been considerable interests in the relationship between tumor cell proliferation and angiogenesis. In human breast carcinomas, it has been reported that angiogenesis is an independent prognostic factor and that proliferating factors are closely related with lymph node status. Therefore, the demonstration of proliferating and angiogenetic factors as well as the tumor blood vessels in human breast carcinoma simultaneously is important for clinical evaluation of breast cancer patients.

CLSM has been an exciting new apparatus because of it's more powerful resolution than conventional light microscopy and the high-performance on 3D optical sectioning. Images taken with CLSM can be digitized and made variable for image analysis manipulation and computer-assisted 3D reconstructions.

In the present study, we employed immunohistochemical comparative 3D visualization of proliferating and angiogenstic factors with microvessels in human breast carcinomas by CLSM.

The tissues from the surgically resected breast carcinoma were fixed in cold 4 % paraformaldehyde(PFA) and routinely processed in paraffin wax.

To facilitate detection of tumor cells at the highest resolution in an athymic nude mouse model system, Balb/c 3T3 cells transformed with the Harvey ras oncogene were transfected with the histochemical marker gene, bacterial lacZ (LZEJ cells). Alternatively, 3T3 cells transformed with the human sis oncogene were transfected with human placental alkaline phosphatase marker gene (APSI cells). Within minutes of subcutaneous injection, these tumor cells could be detected histochemically and the fate of cells followed with time. APSI or LZEJ cells gave very different single-cell morphologies at this site but yielded similar aggregation patterns of cells. Clearance of some cells could readily be detected by diffusion of histochemical product as “curly-haired” organizations of cells condensed into ovoid collections. Expansion of the population occurred with division of many cells in the population, not just one or a few cells. Cell number was quantitated by the development of ultrasensitive luminometry assays for the two histochemical marker enzymes; different kinetics were observed for establishment of LZEJ or APSI cells.

Metastasis is a highly selective nonrandom process favoring the survival of minor subpopulations of metatastatic cells that preexist within the primary tumor. The cellular and molecular mechanisms that regulate the metastasis of tumor cells to specific organs are diverse and both tumor and organ-specific (1). Many examples exist in which malignant tumors metastasize to specific organs. As Paget proposed in 1889 (2), and as our recent biological and molecular evidence demonstrates, the organ microenvironment influences the invasion, survival, growth, and apoptosis of particular tumor cells. This hypothesis explains metastatic colonization patterns that cannot be due to solely mechanical lodgement/anatomical considerations (1). Successful metastasis therefore involves the interaction of tumor cells with a compatible milieu provided by a particular organ environment.

Recent experimental evidence suggests that paracrine stimulation of tumor cells by organ-derived growth factors and cytokines is one mechanism which determines the target organ preference of disseminated cancer cells.

The formation of vascular stroma (angiogenesis) is essential for tumor growth and may also influence invasion and metastasis. Quantitation of angiogenesis may serve as a prognostic marker in certain malignancies and novel therapies targeted at inhibiting angiogenesis may prove to play an important role in control of tumor growth.

In situ hybridization (ISH) studies have helped to define some of the molecular and cellular events responsible for the formation of vascular stroma in malignancy.

Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), is a multifunctional cytokine associated with tumor angiogenesis. Vascular endothelial cells express at least two tyrosine kinase receptors for VPF, flt-1 and KDR. VPF acts as an angiogenic factor in several ways. It is an endothelial cell growth factor, alters endothelial cell gene expression, induces endothelial cell migration, and it is also a potent inducer of microvascular hyperpermeability (50,000 times as strong as histamine).

Non-invasive imaging tools using biosensors and biomarkers for defining the function of living cells and tissues are among rapidly emerging technologies being developed to monitor cellular toxicity mechanisms. Commercially available fluorescence probes and naturally occurring fluorescent molecules can be used to quantitatively monitor a number of functional endpoints in cultured cells. Innovative approaches to exploit the sensitivity, spectroscopy and temporal/spatial resolution properties of fluorescent probes may provide sensitive approaches for analysis of the molecular mechanisms of toxicity in cells exposed to a variety of toxicants. Kinetic analysis of multiple cellular parameters such as intracellular glutathione (GSH) and Ca2+ content, reactive oxygen species (ROS) generation, mitochondrial and plasma membrane potentials, intracellular pH, and gap junction-mediated intercellular communication (GJIC) with cellular component-specific fluorescent probes permits rapid identification of changes in these parameters and the chronology of injury in cells caused by acute toxicant exposure. Cellular signal transduction mechanisms are also likely to be vulnerable to toxic insults.

Plasmodium falciparum, the most malignant human malaria, is responsible for 2-3 million deaths annually. These infections often involve blockage of the cerebral microvasculature by P. falciparum-infected erythrocytes (Fig. 1). This aspect is considered the major factor in the pathogenesis of cerebral malaria.

Upon invasion of the erythrocyte, P. falciparum immediately begins to remodel the infected erythrocyte. The adherence points of infected erythrocytes, termed knobs (Fig. 2 and 3), contain antigenically diverse 200-350kDa surface proteins (PfEMPl; Fig. 4). The PfEMPl variant surface proteins are encoded by a large and extremely diverse family of genes (var), and switches in the expression of var genes account for rapid changes in the antigenic and adhesive properties of P. falciparum-inkcted erythrocytes (2.4% per generation). Switches in the PfEMPl expression may not only affect the phenotype of the parasite strain but may also change its sequestration to endothelial cells. Genetic reorganization in this protein can lead to binding any of the following endothelial cell receptors; ICAM-1, CD36, thrombospondin, chondroitin sulfate (Fig. 5),2 ELAM-1, or VCAM-1.

In the past five years, a case for oxidative stress in the pathogenesis of Alzheimer disease (AD) has been convincingly made. That link owes most of its existence to the microscopic detection of neuronal oxidative damage. Focusing on damage not only has the advantage that stable products are studied, rather than short-lived radicals, but also that damage can be morphologically defined. This latter aspect is critical since biochemical analysis of whole tissue is mostly of the accumulated damage from normal metabolism with aging of long-lived polymers in the extracellular matrix (Figure 1). The importance of damage is that it is the result most likely to be linked to pathology. Driven by the hypothesis that oxidative damage plays a role in the aggregation of insoluble protein in the lesions of Alzheimer disease, neurofibrillary tangles and senile plaques, we developed reagents to detect protein modifications related to glycoxidation, lipid peroxidation, peroxynitrite, free carbonyls and carbonyl-modification (Figure 2).

Proteins of the axonal cytoskeleton, particularly neurofilament and microtubule-associated protein τ, should be particularly sensitive to the effects of oxidative modification due to their high content of lysine, an amino acid that is particularly susceptible to direct oxidization as well as adduction by carbonyls produced from lipid and sugar oxidation. To understand the susceptibility of the cytoskeleton to oxidative modification and whether such modification is related to the physiological function of the cytoskeleton, we undertook a cytological analysis of motor neurons isolated from mouse spinal cord. These neurons contain an abundant axonal cytoskeleton that can be readily analyzed distinct from the cell body. Immunocytochemistry, using antibodies against protein-adducts of the highly reactive lipid peroxidation product, hydroxynonenal (HNE), representing Michael addition or pyrrole formation, revealed that HNE-immunoreactive adducts are found in all axons. This in situ distribution of HNE-adducts is consistent with immunoblots prepared from axons which show selective HNE modification of neurofilament heavy subunit (NFH) but not of other cytoskeletal proteins.