Capacity for differentiation and normalization of tumor cell populations in transplantation into the anterior chamber of the eye. III. Rhabdomyosarcoma A-7 clones

Four clone lines of transplantable cell polymorphic rhabdomyosarcoma A-7 were investigated during transplantation to the subcutaneous connective tissue (SCT) and into eye anterior chamber (EAC). Cell morphology of transplants was studied by light and electron microscopy, the activity of their LDH M- and H-subunits was examined cytochemically, and the quantity of their nuclear DNA--cytophotometrically. In the case of A-7/1, A-7/2 and A-7/3 cell lines of EAC transplants we noticed a decrease in cell element kataplasia levels, differences in LDH M- and H-form ratio, reduction in the karyotype variability. Transplants of A-7/4 clone line were similar in SCT and EAC for all the signs studied. The results obtained show that the transplantable cell polymorphic rhabdomyosarcoma A-7 is heterogeneous for its differentiation and normalizing capacities during EAC proliferation. The data reported elsewhere concerning capability of four lines of murine rhabdomyosarcomas to normalize in EAC are discussed, and some possible mechanisms of this effect are regarded.     

Capacity for differentiation and normalization of tumor cell populations transplanted into the anterior chamber. IV. Ehrlich ascites carcinoma

The activity of LDH M- and H-forms, nuclear DNA contents and of genome mutation rates in the Ehrlich ascitic carcinoma cells were studied after transplantation to the intraperitoneal cavity, subcutaneous connective tissue (SCT) and the eye anterior chamber (EAC). SCT and EAC cultivations demonstrate the increase of M- to H-forms activity ratio due to a lesser H-form activity; so, LDH profiles appear to be associated with such a spontaneously highly differentiated adenocarcinoma of murine breast cancer. SCT and EAC cultivation leads also to changes in the karyotypic structure of cell population--there are no polyploid cells (DNA content is more than 4c). It may result from a sharp fall in genome mutation rates in the Ehrlich ascitic carcinoma clones following EAC proliferation which appears 47 times lower than those during lung proliferation. The data obtained favor a hypothesis that the increase in differentiation level and decrease in tumorigenety of cancer cells during EAC proliferation may be due to selection of the near-diploid cells and to the reduction in genome mutation rates, which in the whole results in decreasing genotypic and epigenotypic variability in tumor cell populations.     

Lactate dehydrogenase activity and its isoenzyme spectrum in the human pancreas in the prenatal period

Lactate dehydrogenase (LDH) activity and the character of its isoenzyme distribution in pancreas of the human embryos and feti of the 5th-13th-week development were studied. It is shown that LDH activity was rather high already in early periods of the embryonic development, peaks of the enzymic activity were observed after 7-8 and 12-13 weeks. The isoenzymic LDH spectrum was characterized by the presence of four isoenzymes: LDH1, LDH2, LDH3, LDH5. Isoenzyme LDH4 was absent in the human pancreas in all the studied periods of embryonic development. The data obtained evidence for intensity of the glycolysis processes at LDH reaction level in the prenatal ontogenesis period and they reflect most probably the processes of development and differentiation ox cellular populations in the given organ.     

Ploidy level and alpha-fetoprotein production in spontaneous murine hematomas in relation to the degree of histological differentiation

Comparative morphological, cytospectrophotometric and immunochemical studies were carried out on 26 hepatocarcinomas of various malignancy excised from 21 F1 (CBA X C57B16) mice. It has been shown that in morphologically similar hepatomas the ploidy level and alpha-fetoprotein production intensity may vary markedly. Highly differentiated carcinomas usually of smaller sizes and homogeneous morphology have the modal class nuclei with tetraploid (6 cases) or octaploid (3 cases) DNA content. In moderately- and low-differentiated carcinomas the diploid class of nuclei significantly increased (in 4 out of 6 tumours). Large tumours morphologically heterogeneous according to the differentiation level had polymodal DNA distributions with predominating tetraploid nuclei (II cases).     

Metamorphosed fibroblasts and their relation to the histogenesis of malignant fibrous histiocytoma in experimental murine model

Malignant fibrous histiocytoma (MFH) is a clinicopathologically established entity, but its histogenesis remains to be clarified. We have reported the existence of a specific cell type, the "fibrohistiocytoid (FH) cells", in various chronic inflammatory tissues. The FH cells are the metamorphosed fibroblasts and we have revealed the morphological resemblance between FH cells and MFH cells. In the present study we carried out some experiments to ascertain whether the FH cells have a possibility of neoplastic potential for the development of MFH in mice. A total of 50 female Balb/c mice treated with a chemical carcinogen, 9,10 dimethyl-1,2-benzanthracene (DMBA), were examined histopathologically from 8 to 22 weeks after the initial treatment. It was found that 1) the chemically induced tumors in the mice resembled human pleomorphic/ storiform variant of MFH and cells from the tumor were transplantable subcutaneously in the back of another mouse, 2) the tumors were composed mainly of malignant FH cells, and there were many benign FH cells and fibroblasts in granulation tissues obtained at the initial stage of the experiment, 3) all DNA histograms obtained from MFHs were aneuploid and granulation tissues were diploid, and 4) benign FH cells in the granulation tissue appeared to have higher DNA synthesis activity than typical fibroblasts on the basis of bromodeoxyuridine (BrdU) labeling and cytofluorometric studies. From these findings, we suggest that the FH cells are not only a merely morphologically changed fibroblast, but also a biologically ominous cell which may contribute to develop MFH in mice.     

Lactate dehydrogenase-elevating agent is responsible for interferon induction and enhancement of natural killer cell activity by inoculation of Ehrlich ascites carcinoma cells into mice

Inoculation of Ehrlich ascites carcinoma cells (EAC) into the peritoneal cavities of outbred ddY mice induced interferon (IFN) in the circulation. The maximum titer (1,280 U) was obtained at 24 hr after inoculation. This induced IFN had the characteristics of type I IFN, i.e., stability at pH2 and lability at 56 C. An increase in natural killer cell (NK) activity was also observed for the first 3 days after inoculation. In addition, plasma lactate dehydrogenase (LDH) activity was elevated in these mice. Inoculation of ascitic fluid or serum of EAC-bearing mice into normal mice increased plasma LDH activity six- to sevenfold over normal levels and elevated activities persisted throughout the life of the mice. These results suggest that the LDH-elevating agent was responsible for IFN induction and for enhancing NK activity. Because lactate dehydrogenase-elevating virus (LDV) can be eliminated from tumor cells by passage in vitro, we attempted to grow EAC in tissue culture for several months and re-examined whether the inoculation of such cells could elevate plasma LDH activity induce IFN and enhance NK activity. The results showed that inoculation of the passaged cells had no effect on these activities in normal mice. Therefore, we concluded that the IFN inducer was LDV which contaminated the EAC and then enhanced the NK activity. N-tropic murine leukemia virus also contaminated EAC, but this virus was not responsible because cultured cells of EAC still shed this virus.     

Duplication of the LDH gene loci by polyploidization in the fish order Clupeiformes

Summary Within the order Clupeiformes, chromosome analysis and DNA measurements have indicated a diploid-tetraploid relationship among closely related species. To confirm the presence of polyploidization at single gene loci we studied the LDH isoenzyme system. The results obtained are in agreement with the hypothesis of polyploidization. While the diploid species show two gene loci for LDH, the tetraploid species exhibit four separate gene loci.In Duarte, supported in part by CA-05138 from the National Cancer Institute, U.S. Public Health Service.     

LDH and LDH Isoenzymes of Synovial Fluid in the Horse

LDH is an intracellular enzyme, which when cells degenerate is released to the extracellular spaces and body fluids. Cells and organs in the mammalian body differ from each other with respect to their LDH isoenzyme patterns. These circumstances have led to the use of LDH isoenzyme determinations in laboratory diagnostic work. In the present investigation total LDH activity and LDH isoenzyme distribution in equine synovial fluid from healthy joints, joints with serous arthritis, osteochondrosis dissecans and arthrosis, were determined. The fluids from the diseased joints differed from normal synovial fluid with respect to total LDH activity, and the different joint diseases each seemed to give rise to a characteristic isoenzyme pattern. In order to examine possible sources of the increased LDH activity and altered isoenzyme patterns, blood plasma, red and white blood cells, synovial membrane and articular cartilage were also studied. It was found that LDH4 and LDH5 were present in high amounts in articular cartilage, and an increase in these isoenzymes was the most characteristic feature in synovial fluid from joints with arthrosis. The results were discussed in view of possible diagnostic value of isoenzyme determinations on synovial fluid.     

Antineoplastic and antibacterial activity of some mononuclear Ru(II) complexes

These ligands (L) show a bidentate behavior, forming octahedral ruthenium complexes. The title complexes were subjected to in-vivo anticancer activity tests against a transplantable murine tumor cell line, Ehrlich's Ascitic Carcinoma (EAC) and in-vitro antibacterial activity against several Gram positive and Gram negative bacterial strains. [Ru(bpy)2(ihqs)]Cl2 and [Ru(bpy)2 (hc)]Cl2 (where bpy = 2,2'-bipyridine, ihqs = 7-iodo-8hydroxy quinoline-5-sulphonic acid and hc = 3-hydroxy coumarin) showed promising antitumor activity. Treatment with these complexes prolonged the life span of EAC bearing mice as well as decreased their tumor volume and viable ascitic cell count. All the tested complexes exhibited mild to moderate antibacterial activity.     

Changes in cytosolic Ca2+ levels correspond to fluctuations of lactate levels in crosstalk of astrocyte-neuron cell lines

Neurons and astrocytes differentially express isoenzymes of lactate dehydrogenase (LDH). The metabolic consequences for the variations in mRNA expression of LDH isoenzyme subtypes in neurons and astrocytes control cerebral vasoregulation. Moreover, cellular signalling consequences for functional neurovascular control may also be dependent on LDH isoenzyme subtype profiles. Initial computer simulations revealed glutamate-induced calcium waves in connected astrocytes, and showed concomitant changes in the expression of nitric oxide synthase (NOS) and lactic acid metabolism. To validate these findings, the nature and extent of glutamate-dependent signalling crosstalk in murine cell lines were investigated through correlated lactate levels and calcium upregulation. Neuro2A and C8D1A cells were separately treated with timed supernatant extracts from each other and their LDH1 and LDH5 isoenzyme responses were recorded. Western blot analysis showed LDH1/LDH5 isoenzyme ratio in the astrocytes to be positively correlated with Neuro2A-derived lactate levels estimated by the amplitude of 1.33-ppm spectral peak in 1H-NMR, and LDH1/LDH5 isoenzyme ratio in neurons is negatively correlated with CSD1A-derived lactate levels. Significant modulations of the calcium-responsive protein pCamKII levels were also observed in both cell lines, particularly correlations between pCamKII and lactate in C8D1A cells, thus explaining the calcium dependence of the lactate response. Together, these observations indicate that lactate is a key indicator of the metabolic state of these cell types, and may be a determinant of release of vasoregulatory factors.     

Capacity for differentiation and normalization of populations of tumor cells transplanted into the anterior chamber of the eye. I. Changes in the pleomorphic rhabdomyosarcomas MKh-III and A-7 of mice

Tumors transplanted into the eye anterior chamber (EAC) were atypic, more differentiated morphologically and with higher activity of H-forms of LDH, compared to the same tumors grown in the subcutaneous connective tissue (SCT). Besides, in the EAC, the tumors were less karyotypically heterogeneous and had lesser mitotic indices, than in the SCT. After cultivation in the EAC, populations of tumor rhabdomyoblasts displayed a decreased transplantability in SCT, i.e. the usual site of their implantation. These results may suggest that the proliferation of tumor cell populations in the EAC, i.e. in the immunologically privileged place, can be accompanied by the reducing of their malignancy up to the total loss of the common tumorigenic ability. Even the cell populations of tumor rhabdomyoblasts from the late stages of tumor progression can undergo such a process of normalization.     

Changes in the lactate dehydrogenase isoenzyme spectrum during T-lymphocyte differentiation

The pattern of lactate dehydrogenase isoenzyme spectrum changes on different stages of T-lymphocyte differentiation was studied An enriched population of stem cells has LDH-5, 4 and 3 isoenzymes, and much less LDH-2 activity. The isoenzyme pattern of thymic cell precursors consists of LDH-5, 4, 3 and 2. All the five LDH isoenzymes were found in cortical thymocytes. Medullary thymocytes reveal LDH-5, 4 and 3 isoenzymes. T-lymphocytes of peripheral lymphoid organs contain mainly LDH-5 and in a lesser degree LDH-4 activity.     

Properties of the testicular lactate dehydrogenase isoenzyme.

1. Studies were carried out with pure lactate dehydrogenase isoenzymes C4 (LDH isoenzyme X), B4, (LDH isoenzyme 1) and A4 (LDH isoenzyme 5) isolated from mouse testis, heart and muscle tissue respectively; with LDH isoenzyme X purified from pigeon testes and with crude lysates of spermatozoa from man, bull and rabbit. 2. LDH isoenzyme X from all species showed greater ability than the other isoenzymes to catalyse the NAD+-linked interconversions of 2-oxobutanoate into 2-hydroxybutanoate and of 2-oxopentanoate into 2-hydroxypentanoate. 3. Mouse LDH isoenzyme X presented the broadest spectrum of substrate specificity. It exhibited very similar Km values for a variety of 2-oxo acids: 2-oxopropanoate (pyruvate), 2-oxobutanoate, 2-oxo-3-methylbutanoate, 2-oxopentanoate, 2-oxo-3-methylpentanoate, 2-oxo-4-methylpentanoate, 2-oxohexanoate and 2-oxo-3-phenylpropanoate (phenylpyruvate). The corresponding 2-hydroxy acids were also readily utilized in the reverse reaction. A strong inhibition by substrate and product was demonstrated for the direct reaction. 4. Intracellular distribution of LDH isoenzyme X was investigated in mouse testes. LDH isoenzyme X activity was located in the fraction of "heavy mitochondria" and in the soluble phase. 5. A possible functional role for LDH isoenzyme X is proposed: the redox couple-2-oxo acid-2-hydroxy acid could integrate a shuttle system transferring reducing equivalents from cytoplasm to mitochondria.     

Morphopathology of mixed sweat-gland tumors

Mixed sweat-gland tumors were studied on 11 biopsies, of which two showed clear-cut morphological features of malignancy. Stress was laid upon the sudoriferous-duct histogenesis of such tumors. Several peculiarities were carefully investigated, especially epithelial proliferation in sheets, cords, tubes, isolated cells and certain epithelial metaplasias correlated with various mesenchymal stroma changes, such as fibrosis, myxoid, chondroid or even osseous changes. Special emphasis was also laid upon the comparative aspects between benign and malignant mixed sweat-gland tumors and other similar body tumors.     

Biologically active sphingolipids in transplantable tumors of different histogenesis

The composition of biologically active sphingolipids in hepatoma 22, breast adenocarcinoma, Lewis lung carcinoma, large intestine adenocarcinoma, cervical carcinoma, and melanomas M3 and B16 was compared to elucidate the similarity and differences in sphingolipids of subcutaneously transplantable murine tumors. The sphingolipid composition of the tumors was found to widely vary. The sphingomyelin, ceramide, glucosyl- and lactosylceramide, and ganglioside GD3 contents in hepatoma 22 are higher than those in normal tissue. No common regularities for tumors of different origin were observed in the ratios of bioeffectors inhibiting proliferation and stimulating apoptosis and bioeffectors stimulating cell growth and survival. However, the Cer/(GlcCer + LacCer) ratios were very low and practically equal in two melanoma strains, which probably indicates the degree of tumor malignancy. The results suggest that the content and composition of sphingolipids in tumors depend on their histogenesis.     

Biologically active sphingolipids in transplantable tumors of different histogenesis

The composition of biologically active sphingolipids in hepatoma 22, breast adenocarcinoma, Lewis lung carcinoma, large intestine adenocarcinoma, cervical carcinoma, and melanomas M3 and B16 was compared to elucidate the similarity and differences in sphingolipids of subcutaneously transplantable murine tumors. The sphingolipid composition of the tumors was found to widely vary. The sphingomyelin, ceramide, glucosyl-and lactosylceramide, and ganglioside GD3 contents in hepatoma 22 are higher than those in normal tissue. No common regularities for tumors of different origin were observed in the ratios of bioeffectors inhibiting proliferation and stimulating apoptosis and bioeffectors stimulating cell growth and survival. However, the Cer/(GlcCer + LacCer) ratios were very low and practically equal in two melanoma strains, which probably indicates the degree of tumor malignancy. The results suggest that the content and composition of sphingolipids in tumors depend on their histogenesis.     

Proliferative characteristics and ploidy of human brain tumors by DNA flow cytometry

We studied nuclear DNA distribution by flow cytometry in 59 human brain tumors. Samples were frozen at -20 degrees C immediately after surgery and unicellular suspensions were obtained with a mechanical dissociation technique. Nuclear DNA was stained with propidium iodide. Normal human brain tissue was used as a diploid reference standard. In 86.3% of benign tumors an unimodal DNA distribution with a DNA index usually within the diploid range was found. Among malignant tumors, 64% had un unimodal DNA distribution with diploid or near-diploid modal DNA content. The remaining 36% showed an additional cell peak with a DNA index ranging from 1.15 to 1.92. The percentage of S-phase cells was higher in malignant (median = 3.8) than in benign tumors (median = 1.9) (p less than .001), without correlation to histological tumor subtype.     

意蜂工蜂、雄蜂和蜂王的LDH和EST同工酶比较

用聚丙烯酰胺凝胶电泳的方法,对意蜂工蜂、雄蜂和蜂王以及两种不同大小幼虫之间的乳酸脱氢酶(LDH)和酯酶(EST)同工酶分别作了比较。结果表明,工蜂、雄蜂和蜂王之间LDH同工酶数目无差异,但活性上存在差异;而酯酶同工酶在幼虫阶段与成虫阶段及工蜂、雄蜂和蜂王之间则有着不同的同工酶谱型。说明意蜂同工酶在种内的表达受到发育程度、进食质量、分化方向等因素的影响。     

Electrophoretic distribution and dissociation into subunits of lactate dehydrogenase derived from human myeloid leukemia cells before and after induction of differentiation

The electrophoretic distribution of lactate dehydrogenase (LDH) isoenzymes, Michaelis constant, reaction with substrate, and dissociation into subunits with guanidine hydrochloride was examined in undifferentiated and differentiated human myeloid leukemia cells. Differentiation was induced with 1/microgram/ml tunicamycin. Undifferentiated cells did not display phagocytic ability, and less than 5% of these cells had Fc receptors. After exposure to tunicamycin for 40 hr, 40% of these differentiated cells had Fc receptors, and 35% showed phagocytic activity after 160 hr. The majority of the LDH activity in the undifferentiated cells was found in fraction 3, and following differentiation almost a 50% reduction in LDH activity was observed in this fraction. In addition, LDH 3 isoenzyme levels were found to be greater in patients containing a high percentage of undifferentiated cells than in patients containing a high percentage of differentiated cells. Differentiated cells displayed LDH isoenzyme fraction pattern, Michaelis constant, and reaction with substrate similar to those found in the normal granulocytes. Differences in the dissociation of LDH into subunits with guanidine hydrochloride were found between undifferentiated and differentiated acute myeloid leukemia (AML) cells. Treatment with 0.75 M guanidine hydrochloride caused complete inactivation of LDH derived from normal differentiated cells, whereas similar treatment caused complete inactivation of LDH derived from AML or normal granulocytes. LDH isoenzymes derived from normal granulocytes and differentiated AML cells were also more sensitive to guanidine hydrochloride depression of fluorescence intensity. The sedimentation constant for single peak LDH at 5.5 M guanidine hydrochloride was calculated as 1.65 sec for differentiated and 1.70 sec for undifferentiated cells. The molecular weight of the polypeptide subunits for undifferentiated cells was 30,000 and for differentiated cells was 39,000. The apparent parallel between leukemic cells after induction of differentiation and normal granulocytes indicates that the leukemic cells retain their maturation potential when exposed to an inducer of differentiation.     

Similar chromosomal abnormalities in several retinoblastomas

Summary The study of banded chromosomes of nine sporadic unilateral retinoblastomas revealed near diploid karyotypes with multiple numerical and (or) structural abnormalities in all tumors. An identical marker i(6p) was noted in cells of the modal class of six retinoblastomas. Extra copies of the short arm of chromosome 6 were observed in seven tumors: +i(6p) in 6 and +6q- in one. Less regular but repeated findings were a loss of one sex chromosome, and markers 1p+ and 17q+. The structure of these markers was not identical in different tumors. Abnormalities of chromosome 13 were not observed in tumor cells, nor in blood lymphocytes stimulated by PHA.