Recombinant human arginase inhibits proliferation of human hepatocellular carcinoma by inducing cell cycle arrest.

Human hepatocellular carcinoma (HCC) has an elevated requirement for arginine in vitro, and pegylated recombinant human arginase I (rhArg-PEG), an arginine-depleting enzyme, can inhibit the growth of arginine-dependent tumors. While supplementation of the culture medium with ornithine failed to rescue Hep3B cells from growth inhibition induced by rhArg-PEG, citrulline successfully restored cell growth. The data support the roles previously proposed for ornithine transcarbamylase (OTC) in the arginine auxotrophy and rhArg-PEG sensitivity of HCC cells. Expression profiling of argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL) and OTC in 40 HCC tumor biopsy specimens predicted that 16 of the patients would be rhArg-sensitive, compared with 5 who would be sensitive to arginine deiminase (ADI), another arginine-depleting enzyme with anti-tumor activity. Furthermore, rhArg-PEG-mediated deprivation of arginine from the culture medium of different HCC cell lines produced cell cycle arrests at the G(2)/M or S phase, possibly mediated by transcriptional modulation of cyclins and/or cyclin dependent kinases (CDKs). Based on these results, together with further validation of the in vivo efficacy of rhArg-PEG against HCC, we propose that the application of rhArg-PEG alone or in combination with existing chemotherapeutic drugs may represent a specific and effective therapeutic strategy against HCC.

"Fathers" and "sons" of theories in cell physiology: the membrane theory.

The last 50 years in the history of life sciences are remarkable for a new important feature that looks as a great threat for their future. A profound specialization dominating in quickly developing fields of science causes a crisis of the scientific method. The essence of the method is a unity of two elements, the experimental data and the theory that explains them. To us, "fathers" of science, classically, were the creators of new ideas and theories. They were the true experts of their own theories. It is only they who have the right to say: "I am the theory". In other words, they were carriers of theories, of the theoretical knowledge. The fathers provided the necessary logical integrity to their theories, since theories in biology have still to be based on strict mathematical proofs. It is not true for sons. As a result of massive specialization, modern experts operate in very confined close spaces. They formulate particular rules far from the level of theory. The main theories of science are known to them only at the textbook level. Nowadays, nobody can say: "I am the theory". With whom, then is it possible to discuss today on a broader theoretical level? How can a classical theory--for example, the membrane one--be changed or even disproved under these conditions? How can the "sons" with their narrow education catch sight of membrane theory defects? As a result, "global" theories have few critics and control. Due to specialization, we have lost the ability to work at the experimental level of biology within the correct or appropriate theoretical context. The scientific method in its classic form is now being rapidly eroded. A good case can be made for "Membrane Theory", to which we will largely refer throughout this article.

Arginine deiminase: a potential inhibitor of angiogenesis and tumour growth.

Hydrolysis of plasma arginine to citrulline by arginine deiminase (ADI) was recently shown to suppress lipopolysaccharide-induced nitric oxide (NO) synthesis. Since arginine is the precursor of NO, and the latter modulates angiogenesis, we explored whether ADI treatment significantly affected tube-like (capillary) formation of human umbilical vein endothelial cells. Inhibition occurred in a dose-dependent manner, both in the chorioallantoic membrane and the murine Matrigel plug assay. Inhibition of angiogenesis by ADI was reversed when a surplus of exogenous arginine was provided, indicating that its antiangiogenic effect is primarily due to arginine depletion, although other pathways of interference are not entirely excluded. Arginine deiminase is also shown to be as a potent inhibitor of tumour growth in vitro as in vivo, being effective at nanogram quantities per millilitre in CHO and HeLa cells. Thus, it could be highly beneficial in cancer therapy because of its two-pronged attack as both an antiproliferative and an antiangiogenic agent.

Arginine deprivation, growth inhibition and tumour cell death: 3. Deficient utilisation of citrulline by malignant cells.

Arginine deprivation causes death of up to 80% of cancer cell lines in vitro, but in the body, citrulline would be available as a convertible source of this amino acid in vivo. Some tumour cell lines, notably the vast majority of melanomas and hepatocellular carcinomas, tend to be deficient in argininosuccinate synthetase (EC 6.5.4.3.), and therefore cannot recycle citrulline to arginine. Argininosuccinate synthetase is present at levels that convert enough citrulline to arginine to allow limited growth in about half of a modest range of malignant cell types analysed in this study. Attempts to rescue cells that are unable to utilise citrulline with the immediate downstream product, argininosuccinate, had very limited success in a few tumour cell lines. Particularly noteworthy is the demonstration that argininosuccinate was totally incapable of rescuing cells that utilise citrulline efficiently, consistent with tight channelling (coupling) of argininosuccinate synthetase and argininosuccinate lyase in the urea cycle. The findings suggest that an excellent opportunity exists for further exploitation of arginine deprivation in the selective killing of tumour cells.

Arginine deprivation, growth inhibition and tumour cell death: 2. Enzymatic degradation of arginine in normal and malignant cell cultures.

Arginase added to culture medium reduced arginine to negligible levels within approximately 6 h, and enzyme activity persisted relatively undiminished for at least 3 days. Human and bovine arginase proved equally effective. The response of normal cells was to enter G1 (G0) arrest, from which most of the cells could be recovered weeks later. In contrast, malignant cell lines treated with unpegylated or pegylated enzyme resulted in cell death on a massive scale within 3 - 5 days, with a very low to negligible percentage of cells (<0.01%) being recoverable on restoration with arginine. Although pegylation resulted in a 40% drop in specific activity, arginase was considerably more stable and remained active for >>8 days. Arginine decarboxylase caused malignant cell arrest at the same units per millilitre as arginase. Its breakdown product, agmatine, was relatively nontoxic in the presence of arginine, but exacerbated cell death above millimolar concentration in its absence. Although ornithine failed to rescue cells from deprivation, citrulline recovered cells in all cases, although less well in fast-growing tumour cell populations, whereas readdition of arginine failed to work unless a complete medium change was given (because of the persistence of the enzymes in the medium catabolising its destruction). The advantages and disadvantages of these two arginine-catabolising enzymes are discussed, and compared with arginine deiminase.

Purification and identification of a human dermal extract component inhibitory to fibroblast proliferation.

It was previously shown that a citric acid buffer extract of human dermis (extract D) inhibited growth of human diploid fibroblasts in monolayer culture (Muir et al., 1997). Further fractionation has shown that the active principle is probably a proteoglycan, and that retention of its inhibitory activity is dependent on the use protease inhibitors throughout the extraction procedure. Elution of extract D from a DEAE-cellulose column produced four major peaks, each of which was subjected to SDS-PAGE as well as being tested for inhibitory activity on the growth of fibroblasts in culture. Peaks III and IV had no inhibitory effect, but peak I contained highly active material. Gels of this peak showed prominent bands of 120 kDa (corresponding to dermatan sulphate proteoglycan II, DS-PG II) and at 45 kDa (corresponding to the core protein). The latter band became more prominent when extract D which had been treated with chrondroitinase ABC was electrophoresed. Their identities were verified by Western blotting. Peak II also contained some slower-acting inhibitory material which has as yet to be identified, but contains little or no protein corresponding to the decorin core-protein. The data indicate that the intact decorin molecule, DS-PG II, is the main inhibitory principle in human skin.

Inhibition of the contraction of collagen gels by extracts from human dermis.

Extracts from the human dermis were prepared and evaluated with respect to their ability to influence fibroblasts to contract collagen gels in vitro. The extract which had the most inhibitory effect on fibroblasts to cause contraction of collagen gels was extract D. It also inhibited fibroblast growth. Inhibition of contraction was not simply related to fibroblast cell numbers and the data suggests a specific effect upon the ability of fibroblasts to cause contraction. Other extracts were without significant effect.

Random walks and cell size.

For many years, it has been believed that diffusion is the principle motive force for distributing molecules within the cell. Yet, our current information about the cell makes this improbable. Furthermore, the argument that limitations responsible for the relative constancy of cell size--which seldom varies by more than a factor of 2, whereas organisms can vary in mass by up to 10(24)--are based on the limits of diffusion is questionable. This essay seeks to develop an alternative explanation based on transport of molecules along structural elements in the cytoplasm and nucleus. This mechanism can better account for cell size constancy, in light of modern biological knowledge of the complex microstructure of the cell, than simple diffusion.

Single amino acid (arginine) deprivation: rapid and selective death of cultured transformed and malignant cells.

The effects of arginine deprivation (-Arg) has been examined in 26 cell lines. Less than 10% of those with transformed or malignant phenotype survived for > 5 days, and many died more rapidly, notably leukaemic cells. Bivariate flow cytometry confirmed that vulnerable cell lines failed to move out of cell cycle into a quiescent state (G0), but reinitiated DNA synthesis. Many cells remained in S-phase, and/or had difficulty progressing through to G2 and M. Two tumour lines proved relatively 'resistant', A549 and MCF7. Although considerable cell loss occurred initially, both lines showed a 'cell cycle freeze', in which cells survived for > 10 days. These cells recovered their proliferative activity in +Arg medium, but behaved in the same manner to a second -Arg episode as they did to the first episode. In contrast, normal cells entered G0 and survived in -Arg medium for several weeks, with the majority of cells recovering with predictable kinetics in +Arg medium. In general, cells from a wide range of tumours and established lines die quickly in vitro following -Arg treatment, because of defective cell cycle checkpoint stringency, the efficacy of the treatment being most clearly demonstrated in co-cultures in which only the normal cells survived. The findings demonstrate a potentially simple, effective and non-genotoxic strategy for the treatment of a wide range of cancers.

Single amino acid (arginine) restriction: growth and death of cultured HeLa and human diploid fibroblasts.

Requirements for arginine are different from leucine for the growth of HeLa cells in monolayer and suspension culture. Cells grow increasingly more slowly as arginine levels fall below millimolar. Most cells died at 10(-5) M in static cultures, but could be sustained in perfused cultures, but at 10(-6) M neither perfusion nor increased volume in static cultures compensated. Cell died within 3-4 days in 10(-6) M in the same manner as those in complete arginine deprivation, i.e. considerably faster than with leucine deprivation. Arginine restriction produced by arginase or arginine decarboxylase addition to culture medium gave similar results. Citrulline substituted for arginine, but ornithine and polyamines did not. Arginine was depleted 3-4 times faster from the medium than other amino acids, <5% being consumed in protein synthesis, and arginine released by protein turnover was less efficiently reutilised than leucine. Deprivation reduced protein and DNA syntheses, greatly extended S-phase and protracted the cell cycle in HeLa cells for more than leucine deprivation. The inability of the cells avoid reinitiation of S-phase resulted in their proliferative impetus driving them into an late cycle (premitotic) death. In contrast, normal human diploid fibroblasts reached quiescence with little delay and survived for >11 days. Arginine deprivation is discussed as a selectively means of tumour cell destruction.

Single amino acid (arginine) deprivation induces G1 arrest associated with inhibition of cdk4 expression in cultured human diploid fibroblasts.

Withdrawal of a single amino acid (arginine) from freely cycling early passage primary human fibroblasts caused a halt to proliferation, characterized by an accumulation of cells in the G1 phase of the cell cycle. This arrest was accompanied by the suppression of cyclin D1- and cyclin E-associated kinase activities and the appearance of hypophosphorylated retinoblastoma protein. Arginine-deprived cells remained viable for in excess of 4 days and could be made to synchronously reenter the cell cycle by restoration of the amino acid, with kinetics characteristic of exit from a quiescent state. Stimulation of cells arrested by serum withdrawal did not result in S-phase entry when arginine was omitted from the culture medium. Although cyclin D1 accumulated on normal schedule, cdk4, which increased following restimulation in amino acid-replete medium, was not induced when arginine was absent. These results suggest that arginine deprivation-in common with other "suboptimal" conditions-inhibits the passage of normal human cells through the restriction point and implicate cdk4 as the key regulatory element in amino acid-sensitive cell cycle control.

Diffusion theory in biology: a relic of mechanistic materialism.

Diffusion theory explains in physical terms how materials move through a medium, e.g. water or a biological fluid. There are strong and widely acknowledged grounds for doubting the applicability of this theory in biology, although it continues to be accepted almost uncritically and taught as a basis of both biology and medicine. Our principal aim is to explore how this situation arose and has been allowed to continue seemingly unchallenged for more than 150 years. The main shortcomings of diffusion theory will be briefly reviewed to show that the entrenchment of this theory in the corpus of biological knowledge needs to be explained, especially as there are equally valid historical grounds for presuming that bulk fluid movement powered by the energy of cell metabolism plays a prominent note in the transport of molecules in the living body. First, the theory's evolution, notably from its origins in connection with the mechanistic materialist philosophy of mid nineteenth century physiology, is discussed. Following this, the entrenchment of the theory in twentieth century biology is analyzed in relation to three situations: the mechanism of oxygen transport between air and mammalian tissues; the structure and function of cell membranes; and the nature of the intermediary metalbolism, with its implicit presumptions about the intracellular organization and the movement of molecules within it. In our final section, we consider several historically based alternatives to diffusion theory, all of which have their precursors in nineteenth and twentieth century philosophy of science.

Length control of primary cilia: analysis of monociliate and multiciliate PtK1 cells.

Lengths of primary cilia in cultured PtK1 cells, on both a population basis and within individual (multiciliate) cells, have been compared. The latter examines the degree of discrepancy between cilia arising within the same cell and using a common precursor pool, on the hypothesis that a better correlation would be expected between cilia issuing from the same centrosome than between those in the population in general. To obtain accurate et plentiful measurements, a 'flow-fixation' technique was devised, which flattens the long primary cilia of cultured PtK1 cells (a kidney epithelial cell line from Potorous tridactylus, the kangaroo rat), prior to immunostaining with an antibody directed against detyrosinated tubulin (ID5). Comparisons of the flow-fixed measurements with a through-focus procedure for upright cilia in conventionally fixed cultures showed reasonable agreement, but not as closely as with measurements made on the living cells using the edge-on method of Roth et al. (J. Cell Sci. 89 (1988) 457). The incidence of multiciliation of confluent PtK1 cells cultures was approximately 5%, of which the majority were biciliates. Although shaft length in general varied considerably, biciliates and multiciliates showed a greater internal consistency, with discrepancies of < 25% in 70% of the cases. On both accounts, this consistency is far poorer than in, for comparison, Chlamydomonas, where its two flagella were < 5% different in length and within 10% tolerance throughout the whole population. Thus, length of primary cilia in PtK1 cell populations is considerably less stringently controlled than in PtK1 cells bearing 9 + 2 cilia, but those issuing from a single multiciliated cell tend to show better correspondence.

Cytoplasmic vacuolation, adaptation and cell death: a view on new perspectives and features.

The review focuses on a widely-observed morphological phenomenon, a unique class of cytoplasmic vacuolation, found in cultured (mammalian) cells. This vacuolation is quite distinct from autophagosomal and heterophagosomal, i.e. excessive lysosomal vacuolation, and occurs in most cell types spontaneously or via a wide range of inductive stimuli. Apart from vacuolation arising artefactually (usually due to poor fixation), spontaneous vacuolation occurs in individual or small clusters of cultured cells without apparent change in their local environment, while neighbouring cells remain completely unaffected. Since spontaneous vacuolation is unpredictable, the process of vacuolation--or 'vacuolisation'--('Vacuolation' is the state of being with vacuoles; 'vacuolisation' therefore implies the process of becoming vacuolated. However, only the quicker term vacuolation will be used throughout this review to refer to the process of vacuole development.) induced experimentally, and hence relatively reproducibly by a range of substances and disturbances, offers an experimental approach which should give further insight into its physiology and pathophysiology. Unfortunately, our knowledge here remains woefully inadequate compared with the purely morphological aspects of the phenomenon. Vacuolation following disturbances could have an underlying common mechanism; however, a review of the literature suggests that this is not the case, and that it occurs via several different pathways, involving many different cell organelles and structures. All cells appear to retain the capacity to vacuolate for some physiological purpose, and it can be a permanent feature in many cell types, particularly 'lower' organisms and plants. Vacuolation in cells is generally seen as an adaptive physiological response, presumably for 'damage limitation', but very little is known about the intracellular homeostatic mechanisms which operate to restore the status quo. Where damage limitation fails, cells usually die quickly, but no clear evidence has been found that this is in any way 'programmed'. It is argued that the demise which occurs via the vacuolation route may, in fact, be a distinct form of cell death which is difficult to fit into the conventional lytic and apoptotic modes.

On the vital role of fluid movement in organisms and cells: a brief historical account from Harvey to Coulson, extending the hypothesis of circulation.

The importance of the orchestrated movement of substrates over multicatalytic enzymes, topographically arranged for maximum efficacy for metabolic function, means that the organization of the body right down to the subcellular level is based upon the principle of perfusion for most normal living processes. The history of the development of this idea, which is contrary to the received wisdom arising and still persisting since the early days of biochemistry, will be followed from the time when the overall need for a circulation at the gross level was fully appreciated, extending the argument through a historical perspective to include the control of flow within the individual cell. Life, seen from this new perspective, seems to be inherently dependent upon this principle to such an extent that it leads to a clearer distinction between the living and the dead, and provides a framework for more critical experiments concerning control mechanisms in the cell.

Molecular subdivision of the cortex of dividing Tetrahymena is coupled with the formation of the fission zone.

In contrast to a mitotic-spindle-associated bipolar cytokinesis, the cytokinesis of polarized ciliates is preceded by a reorganization of the cortex into dual metameric patterns for prospective daughter cells and then separated by a transverse fission line. This study concerns relations between the generation of cortical metamery and the formation of the fission line in an amicronuclear (i.e., without mitotic spindle) ciliate, Tetrahymena pyriformis. The fission line appears in the division of T. pyriformis as a transverse line formed by equatorial gaps in the meridional ciliary rows, with the second oral structure (OA2) formed posterior to it. It was found that the metamery of cortical morphogenesis is expressed by the appearance of increased MPM2 antibody binding in dividing cells in an apical area and posterior to the fission line gaps, including patterned changes of this binding in both oral apparatuses (OA1 and OA2), and by a reciprocal decrease of binding of an anti-epiplasm antibody. These tested antigens are localized to different cortical structures, but in predividing cells both uniformly show formation of the fission line contrast of labeling. A serine/threonine kinase inhibitor, 6-dimethylaminopurine (6-DMAP), was applied to dividing T. pyriformis at specific stages: (1) if 6-DMAP was added to early dividing cells, it prevented cells from initiating cytokinesis. (2) If 6-DMAP was added to cells at stages close to the physiological transition point of cell division, it yielded either (i) a partial formation of the fission line on the ventral side, combined with modified growth of undivided cortex adjacent to the fission line, with abnormal cytokinesis, or (ii) variable anterior displacement of the complete fission line, which contracted slowly but uniformly. (3) If 6-DMAP was applied during cytokinesis, it did not delay cell division, but daughter cells become abnormal and underwent an incomplete oral reorganization. These results suggest that the generation of metamerism in the cortex of T. pyriformis involves differentiation of the asymmetric fission zone. At least four stage-dependent 6-DMAP-sensitive effects jointly control the progress of cell division and the mutual spatial relations between the generation of metamery and the appearance, completeness, and position of the fission zone in the cortex of polarized T. pyriformis.

Origins of signalling and memory: matters of life versus death.

This review focuses on the principles in cell-cell communication and cellular ability to respond to external chemical changes which have been so crucial for the development of life on planet Earth. We now know that the capacity of free-living organisms which evolved more than a billion years ago to respond to intercellular signal molecules, originating either from themselves or from other sources in their vicinity, is so similar possibly even more sophisticated - to that of the cells in our own body, and these findings have had a major impact on our struggle to understand how life has evolved and how it can be maintained. Attention is drawn to the very important topic of mechanisms in cell death, being seen as an aggressive and very powerful instrument in the continuance of life and ability of life to proliferate into a plethora of new species, and use insulin-related material as our paradigm. Such signal molecules (hormones) may have played a major role in cellular maintenance throughout evolution.

Helical extrusions at the nuclear envelope: possible involvement in nucleocytoplasmic trafficking.

The existence of intracytoplasmic helical inclusion 24 h after serum stimulation of 3T3 cells which had previously been serum deprived (0. 2%) for 2 days has been previously reported. Further evidence of their relationship with nuclear pores has been found, and their involvement in nucleocytoplasmic transport seems more probable. Helical formations resembling those originally seen have been found seemingly extruding from nuclear pores into the cytoplasm. However, it is unlikely that these structures contribute significantly to the translocation of material from the nucleus to the cytoplasm on a regular basis because their occurrence is rare. An association of the helical complexes with microfilaments has also been observed. Their relationship to annulate lamellae remain enigmatic.

An in vitro model of angiogenesis: basic features.

This report describes a model of angiogenesis which develops in admixtures (co-cultures) of human umbilical vein endothelial cells (HUVEC) and human diploid fibroblasts of dermal origin from adult patients. The system does not require the addition of further growth factors other than those normally present in endothelial growth medium (EGM), nor matrix proteins, and cell growth and proliferation are allowed to occur in a standard low (2%) concentration of fetal calf serum. Angiogenesis was specifically stimulated in response to vascular endothelial growth factor (VEGF), resulting in an increased development of structures resembling a microvasculature bed. Alternatively, angiogenesis was inhibited by addition of an excess of neutralising anti-VEGF antibodies, and the anti-angiogenic drugs such as suramin. We briefly show that stimulatory and inhibitory activities can be easily and quickly quantified by image analysis. Tubule formation was confirmed by confocal and electron microscopy, and the development and disposition of these structures within the co-cultures has been analysed immunochemically to show expression of specific endothelial cell determinants, such as PECAM-1. On this and a number of other criteria, the findings validate this in vitro process as a model of in vivo angiogenesis that can be quantified to assay stimulatory and inhibitory agents, signals and drugs.