A role of the cancer cell membrane fluidity in the cancer metastases: an ESR study.

AH66F or Yoshida sarcoma (YS) cells were transplanted intraperitoneally into male Donryu rats. Cancer cells obtained from ascites were suspended in saline solution (10(7) cells/ml) after washing. Then, 0.1 ml of each suspension obtained from both strains was injected into the tail vein of 5 rats, respectively. Each metastatic nodule, 1 mm or less in a diameter, thus obtained was then injected into the peritoneal cavity in which these metastatic cells come to free. After 10 days, cancer cells obtained from each ascites were suspended in phosphate buffered saline (Ca2+ and Mg2+ free, pH 7.2) after washing. Each suspension (10(7) cells/ml) was violently vibrated with a definite amount of 5-doxyl stearic acid and spin labeling of cancer cell membrane was done. Furthermore, each specimen thus obtained was subjected to the electron spin resonance (ESR) measurement and the order parameter was determined from the spectra. In both YS and AH66F strains, the cell membrane fluidity of the metastatic cancer cell was increased at each temperature measured from 5 degrees C through 35 degrees C. The results obtained here suggest that the change of the cell membrane fluidity of cancer cell is closely related with the cancer metastases.

The use of scanning electron microscopy for investigations into the three dimensional organisation of the interphase nucleus.

In an attempt to elucidate three dimensional information on the organisation of the nucleus, nuclei have been extracted from ascites tumour cells or tissue culture cells by a variety of biochemical techniques, and prepared for high resolution scanning electron microscopy using an osmium-thiocarbo-hydrazide infiltration procedure which has previously proved successful for analysis of chromosome structure. Nuclei were prefixed with either Methanol-Acetic acid, glutaraldehyde or formaldehyde and then extracted by a variety of detergents with the aim of a 'biochemical dissection' of their outer elements to allow surface visualisation of the nuclear lamina. Continued extraction removed all aspects of the nuclear periphery and allowed direct visualisation of the in situ organisation of the chromatin, apparent as at least two levels of supercoiling.

The possible role of stromal influences in tumor cell heterogeneity. Histology and fibronectin pattern of the Yoshida sarcoma of the rat.

Yoshida tumors implanted subcutaneously in the rat are conspicuously heterogeneous. A definite zoning can be discerned. Cell nuclei range from uniform epitheloid (type I) via pleomorphic (type II) to compact (type III). Type III is seen to detach at the rim of the tumor and to infiltrate the dermis. Type I is characterized by positive pericellular fibronectin staining, type II and III are fibronectin-negative. A marked spatial relationship was seen between types II and III on one hand and the presence of collagenous stroma on the other. It is conjectured that stromal components play a role in tumor promotion or progression.

[Cancer metastasis and blood vessels].

The ultrastructural features on interaction of tumor cells and blood vessels in the process of hematogenous metastasis formation by Yoshida sarcoma, 5 strains of Yoshida rat ascites hepatoma, murine B16 melanoma variant sublines, and others have been described and illustrated. Intravasation and extravasation of tumor cells appeared to be similar cell biological phenomenon, in which two different ways were involved: 1) tumor cells migrated through the pores in the vascular walls which were produced by direct actions of tumor cells, and 2) endothelial cell cytoplasms enclosed the tumor cells neighboring to the vascular walls and as a result intravascular or extravascular tumor cell movement occurred. Tumor cells arrested in a target organ were in close contact with vascular endothelial cells and further with basement membrane when the endothelial linings were removed. In some regions we found junction-like structures between these cells. This appeared to be corresponding to the lodgement of tumor cells in the target organ. Extravasated tumor cells produced three different features of metastatic lesions in the combination of tumor strain and the organ affected; formation of tumor nodules accompanying neovasculature, spreading of tumor cells along the perivascular tissue of an organ, and diffuse infiltration of tumor cells in an organ. It has been discussed that these features in every process mentioned above are determined by specific interaction of tumor cells and host cellular components, especially blood vessels including tumor neovasculature.

Selective targeting of magnetic albumin microspheres to the Yoshida sarcoma: ultrastructural evaluation of microsphere disposition.

Magnetic albumin microspheres (1 micron average diameter) were selectively targeted to subcutaneous solid Yoshida sarcoma tumors (average size 450 mm2) in Holtzman rats. This was accomplished by placing an external magnet adjacent to the tumor while the microspheres were infused. Microspheres contained ultra-fine particles of Fe3O4 and no drug (placebo). Placebo microspheres were used due to the previously demonstrated rapid tumoricidal effect of targeted low-dose doxorubicin microspheres. Animals were killed 10 min, 60 min, 30 min, 24 hr and 72 hr after microsphere administration and tumors were examined by transmission electron microscopy to determine the in vivo disposition of the magnetically targeted microspheres. Using placebo microspheres, we have demonstrated microspheres endocytosed in endothelial cells as early as 10 min after infusion. By 30 min microspheres can be seen in the extravascular compartment, sitting adjacent to tumor cells and occasionally in tumor cells. By 24 hr the majority of microspheres have been endocytosed by tumor cells. Microspheres were still observed within tumor cells as late as 72 hr after administration. The rapid extravasation and cellular uptake of magnetically focused microspheres explains the extremely rapid tumoricidal effect previously observed when doxorubicin-containing microspheres were targeted to the tumor.

The number of nucleolar silver-stained granules of active nucleolus organizer regions in mitotic and interphase cells of rat Yoshida and Zajdela ascitic tumors.

To provide more information on the number of silver-stained granules (SSGs) of active mitotic nucleolus organizer regions (NORs) as well as interphase nucleoli, rat Yoshida ascitic sarcoma and Zajdela ascitic hepatoma cells were studied by means of standardized one-step and two-step silver-staining procedures. The number of SSGs of mitotic active NORs was relatively constant and corresponded to the number of active NORs of animals bearing the tumors investigated. Some anaphases and telophases were "asymmetric", i.e. chromosomal figures (future nuclei) in one and the same cell contained different number of SSGs (active NORs). The incidence of such asymetric anaphases and telophases was higher in aneuploid (hypoploid) Zajdela hepatoma than in euploid (diploid) Yoshida sarcoma cells. In the interphase, the number of SSGs was low in small or large cells with distinct chromocenters or chromosomes condensation presumably representing postmitotic and premitotic cells. In contrast, the highest number of SSGs was noted in nucleoli of large cells which were usually characterized by a fine chromatin structure.

A further study on the number of silver stained granules of active nucleolus organizer regions in mitotic Yoshida and Zajdela rat experimental tumor cells.

Yoshida ascitic sarcoma and Zajdela ascitic hepatoma were investigated in Norway rats to provide more information on active nucleolus organizer regions (NORs) represented by silver stained granules (SSGs). Diploid Yoshida sarcoma cells were characterized by the presence of 6 (3 doublets) of SSGs in the metaphase and 3 + 3 SSGs in future daughter nuclei in the anaphase or telophase. In contrast Zajdela hepatoma cells (frequently hypoploid) possessed 6 SSGs less frequently in the metaphase and the number of anaphasic or telophasic cells with 3 SSGs in future daughter nuclei was also reduced. The number of SSGs in future nuclei of anaphasic or telophasic Yoshida sarcoma cells was usually the same, i.e. such anaphases or telophases were symetric. In Zajdela hepatoma cells the number of symetric anaphases and telophases was substantially reduced in favor of asymetric anaphases or telophases which contained different number of SSGs in future nuclei.

Further studies on the cytochemistry of the standardized silver staining of interphase nucleoli in smear preparations of Yoshida ascitic sarcoma cells in rats.

Silver staining procedure for the selective demonstration of nucleolar silver stained granules (SSG) and for the simultaneous demonstration of SSG and nucleolar silver stained matrix (SSM) were studied in smears of rat Yoshida sarcoma cells. The successful results of these procedures depend mainly on the quality of silver nitrate and formaldehyde. However, both chemicals can be easily standardized and stabilized disregarding their origin and batch. In standardized procedures (one-step procedure for the selective demonstration of SSG and two-steps procedure for the simultaneous demonstration of SSG and SSM) the silver is apparently bound to acidic groups of proteins of SSG and SSM. The proteins of SSG and SSM seem to be different but both belong to the group of acidic non-histone proteins. According to the results of digestion experiments a possibility also exists that the acidic proteins of SSG may be associated with DNA. The identification of SSG visualized by described standardized procedures was determined not only by cytochemical extraction tests but also by biological experiments. The latter demonstrated that the number of SSG in Yoshida sarcoma cells decreases after treatment of experimental animals with actinomycin D and therefore depends on the state of the nucleolar RNA synthesis.

Nucleolar silver stained granules in rat Yoshida sarcoma cells after RNA synthesis inhibition.

The number of nucleolar silver stained granules representing nucleolus organizer regions in interphase nuclei was studied in Yoshida ascitic sarcoma cells without and after the inhibition of the nucleolar RNA synthesis with actinomycin D or cyclophosphamide. The results demonstrated that the number of nucleolar silver stained granules decreased after the inhibition of the nucleolar RNA synthesis with these drugs disregarding the mode of their action. In addition, the decrease of nucleolar silver stained granules in number produced by actinomycin D was dose dependent. Similarly, the number of nucleoli without silver stained granules increased depending on the dose of the administered actinomycin D.

Criteria for the selection of second-generation platinum compounds.

Our selection of a potential second-generation platinum compound began with an initial short list of 8 compounds selected on the basis of antitumour and toxicity studies in mice. We now report further, more detailed investigations of the renal toxicity and antitumour activity of one of these compounds, cis-dichloro trans-dihydroxy bis isopropylamine platinum (IV) (CHIP), in comparison with cis-dichloro diammine platinum (II) (Neoplatin). CHIP was a more effective anti-tumour agent against both alkylating-agent sensitive and resistant strains of the Yoshida sarcoma (YSS and YSR respectively) than was Neoplatin. In addition CHIP produced negligible kidney toxicity as measured by blood urea levels. We have also compared the effects of these two drugs on nuclear-protein phosphorylation, in an attempt to gain insight into their molecular mode of action. Both Neoplatin and CHIP induced increased nuclear-protein phosphorylation in the YSS tumour cells, and loss of condensed chromatin. However, CHIP also induced increased nuclear-protein phosphorylation and loss of condensed chromatin in the YSR tumour cells. These changes correlated well with cell death. In addition Neoplatin, but not CHIP, treatment caused increased nuclear-protein phosphorylation in kidney tissues. This correlated with kidney damage as measured by blood urea levels. These selection criteria suggested that CHIP would be a more selective antitumour agent than Neoplatin, and will provide a basis for its comparison with the other 7 compounds.

Behavior pattern of rat ascites tumor cells arrested in liver sinusoids: an electron microscopic study.

The behavior pattern of tumor cells in the development of hematogenous liver metastasis by Yoshida sarcoma and two strains of rat ascites hepatoma, AH7974F and AH13, in Donryu strain rats was investigated by electron microscopy. Tumor cells of every strain in liver sinusoids stretched their cytoplasmic protrusions toward the vascular wall and were actually in contact with the endothelial cells with their tips to form various types of junction-like structures. The most distinct one was characterized by parallel and straight arrangement of the opposing cell membranes across a gap of about 100 A. The endothelial discontinuity was easily produced by the projection of cytoplasmic processes and the compression of tumor cells. Through the endothelial defect, tumor cells of Yoshida sarcoma and AH13 migrated locomotively into the dilated spaces of Disse or directly toward the hepatic cells, while AH7974F cells were frequently covered by elongated endothelial cytoplasm. Destruction of the hepatic cells by AH7974F and AH13 cells was found to begin with the insertion of their cytoplasmic processes into the coated vesicles in the hepatic cells. From these investigations, the particular interaction between tumor and normal cells seems to play an important role in the development of liver metastasis by these tumors.

[Ultrastructural changes in the liver of rats with a Yoshida tumor under exposure to cyclophosphamide]. / Ul'trastrukturnye izmeneniia pecheni u krys s opukhol'iu Ioshia pri vozdeistvii tsiklofosfamidom.

Sixteen Wistar line rats with Joshida tumor were given cyclophosphamide either daily or with two-day intervals. The changes observed were mostly those related with the Golgi complex apparatus, lysosomes, biliary canaliculi as well as hyaloplasma. The degree of pronouncement of these changes was dependent on the duration of cyclophosphamide administration. The ultrastructure of biliary canaliculi changed according to the type, observed in intrahepatic cholestasis, makes it mandatory to be careful while using cyclophosphamide in patients with the cholestatic syndrome.

Some effects of chlorambucil on nuclear protein phosphorylation in the Yoshida ascites sarcoma.

The ability of nuclei, isolated from Yoshida ascites sarcoma cells, to phosphorylate nuclear proteins in the presence of [gamma-32P]ATP has been investigated. Comparisons were made between a strain sensitive to the effects of the alkylating agent, chlorambucil, with a corresponding resistant strain both before and after drug-treatment of tumour-bearing animals. There was no gross quantitative differences between the drug-sensitive and-resistant untreated cells but treatment resulted in increased levels in the sensitive strain. Qualitative differences were seen before treatment in the phosphorylation pattern of the tris-saline-soluble nuclear sap fraction. The high molecular weight species in the fraction from sensitive cells showed phosphorylations which were absent, or present at very low levels, in the corresponding fraction from drug-resistant cells. Changes were observed in the tris-saline-soluble and non-histone protein fractions from drug-sensitive cells following treatment of tumour-bearing animals. Only minor alterations in patterns of phosphorylation were seen in fractions from drug-resistant cells.

[Cytomorphological and enzyme histological studies of bilaterally inoculated Dibromdulcit-sensitive and-resistent Yoshida tumors]. / Cytomorphologiai és enzimhisztokémiai vizsgálatok bilaterálisan oltott Dibromdulcit-érzékeny és rezisztens Yoshida-tumorokon

In the bilaterally growing DBD sensitive Yoshida tumours deformed nuclear divisions necrosis of the majority of the tumour and appearance of giant cells can be observed due to the single administration of the LD50 of the preparation. The histochemical activity of the LDH the activity alcalyc Adenosine triphosphatase and the nonspecific alcalyc phosphatase become negative while the acidic phosphatase's activity does increase after the administration of the LD25 of the preparation appearance of giant cells are very marked more than 60% of tumours cells are polynuction. The activity of the alcalyc ATP-ase and nonspecific phosphatase decrose's after a transitory increase and become negative while the acid phosphatase's activity increases. In the case of the DBD resistant tumours the morphological and histochemical alternations due to LD50 of the preparation are much slighter and their timecourse is shorter. No morphological and histochemical changes are observed after the administration of LD25 of the preparation.