Transporting cells over several days without dry-ice.

This paper describes a simple, hazard-free and inexpensive procedure that allows researchers to send cultured cells across the globe at ambient temperatures. The method enables transit of up to 2 weeks without compromising cell recovery. Its use will assist collaborators in distant laboratories to exchange cells without using dry-ice.

Preliminary efficacy, safety, pharmacokinetics, pharmacodynamics and quality of life study of pegylated recombinant human arginase 1 in patients with advanced hepatocellular carcinoma.

This study was designed to evaluate the efficacy, safety profile, pharmacokinetics, pharmacodynamics and quality of life of pegylated recombinant human arginase 1 (Peg-rhAgr1) in patients with advanced hepatocellular carcinoma (HCC). Patients were given weekly doses of Peg-rhAgr1 (1600 U/kg). Tumour response was assessed every 8 weeks using RECIST 1.1 and modified RECIST criteria. A total of 20 patients were recruited, of whom 15 were deemed evaluable for treatment efficacy. Eighteen patients (90%) were hepatitis B carriers. Median age was 61.5 (range 30-75). Overall disease control rate was 13%, with 2 of the 15 patients achieving stable disease for >8 weeks. The median progression-free survival (PFS) was 1.7 (95% CI: 1.67-1.73) months, with median overall survival (OS) of all 20 enrolled patients being 5.2 (95% CI: 3.3-12.0) months. PFS was significantly prolonged in patients with adequate arginine depletion (ADD) >2 months versus those who had ≤2 months of ADD (6.4 versus 1.7 months; p = 0.01). The majority of adverse events (AEs) were grade 1/2 non-hematological toxicities. Transient liver dysfunctions (25%) were the most commonly reported serious AEs and likely due to disease progression. Pharmacokinetic and pharmacodynamic data showed that Peg-rhAgr1 induced rapid and sustained arginine depletion. The overall quality of life of the enrolled patients was well preserved. Peg-rhAgr1 is well tolerated with a good toxicity profile in patients with advanced HCC. A weekly dose of 1600 U/kg is sufficient to induce ADD. Significantly longer PFS times were recorded for patients who had ADD for >2 months.

Protein balance: a fundamental question of cell biology needing reappraisal.

Although protein synthesis and protein degradation are two independent processes that are firmly regulated, how they maintain a balance of protein in the non-growing cell remains to be established. In work in the 1980s, the author suggested a self-regulating mechanism. However, experimental work on this interesting and fundamental problem is needed for a better understanding of 'protein balance' in cells.

Characterization and modulation of fibroblast/endothelial cell co-cultures for the in vitro preformation of three-dimensional tubular networks.

Various assays of different complexity are used in research on angiogenesis in health and disease. The results of these assays increasingly impact the field of tissue engineering because preformed microvascular networks may connect and conduct to the vascular system of the host, thereby helping us to support the survival of implanted cells and tissue constructs. An interesting model that supports the formation of EC (endothelial cells) tubular structures in vitro is based on co-culturing them with fibroblasts. Our initial multilayer approach was recently transferred into a three-dimensional spheroid model using HUVEC (human umbilical vein endothelial cells) as model cells. The aim of the present study is to further characterize, extend and validate this fibroblast/EC spheroid co-culture system. We have evaluated the model with a maximum size of 600-650 µm attained on day 3 from inoculation of 4×104 fibroblasts with 1×104 EC. Cell count and spheroid diameter significantly decreased as a function of time, but the EC network that developed over a period of 14 days in culture was clearly visible and viable, and central cell death was excluded. We successfully included HMVEC (human microvascular endothelial cells) of dermal origin in the system and replaced FBS (fetal bovine serum) with human AB serum, which positively impacted the EC network formation at optimized concentrations. The need for exogenous growth factors [VEGF (vascular endothelial growth factor), EGF (epithelial growth factor), bFGF (basic fibroblast growth factor) and IGF-1 (insulin-like growth factor-1)] routinely added to classical EC media was also assessed. The behaviour of both fibroblasts and EC in response to a combination of these exogenous growth factors differed critically in fibroblast/EC spheroid co-cultures compared with the same cells in the multilayer approach. VEGF was the most relevant exogenous factor for EC network formation in fibroblast/EC multilayers, but was ineffective in the spheroid system. IGF-1 was found, in general, to be dispensable; however, while it had a negative impact on EC networking in the presence of bFGF and EGF in the multilayer, it did not in the spheroid approach. We conclude that the critical determinants of EC network formation and cell survival are not universal, but have to be specifically optimized for each culture model.

Primary cilia: turning points in establishing their ubiquity, sensory role and the pathological consequences of dysfunction.

For over 20 years it has finally become accepted that primary cilia are without doubt important cellular organelles, involved in signalling both intrinsically and extrinsically. The consequences of their agenesis, incorrect assembly and dysfunction only began to be fully appreciated after 2000, although this had been demonstrable over the previous two decades. Before 1980, biologists at large thought the organelle rudimentary or vestigial; how a well-developed cilium could be so slated beggars belief. Many pathological conditions have implicated the primary cilium as either a major or contributing factor, ranging from kidney malfunction (e.g. polycystic kidney disease) to mental aberrations. However, the questions of how the recognition of their prevalence, their sensory function, and their pathological involvement finally emerged as substantiated and verifiable facts needs to be addressed because what happened before the 1980s, and then notably between 1980 and 2000, can help guide research towards answering further questions on these issues. Here the intention is to focus on the salient findings (the turning points) that brought about changes in our knowledge of primary cilia. The literature on them is growing fast, with the total moving towards 20,000 reports, of which > 60% have been published in the last decade. PubMed indicates that nearly 1000 papers were published in 2020 alone. We also have to appreciate that the primary cilium can assume many different forms, each of which means that there must be many genes responsible for their development and final structure. This also suggests that there are many more functions than are currently known in both their sensory reception and signalling properties, probably for many highly specialised purposes. Malfunctioning in any of these roles will undoubtedly uncover further pathological conditions.

Reflections on the responsible conduct of cancer research.

Most cancer researchers regularly practice the responsible conduct of research (RCR) without consciously considering it. As professional scientists, we simply do what we are trained to do. However, as we train a new generation of cancer researchers in our laboratories, we must be vigilant against undue complacency. In an age when misconduct in research is receiving more media attention than ever before, we should periodically take a moment of pause and reflect upon the meaning and practice of responsibly conducting research. Rather than meeting minimum standards in a compliance-driven manner, we should practice forethought and periodically consider how we can improve. We, as leaders in cancer research, must then push our peers to do the same. By embedding RCR into the culture of cancer research through a multilayer approach, including regular assessment at the levels of individual research groups, departmentally, and institutionally, we will become a model discipline in the responsible conduct of research.

Another decade of advances in research on primary cilia, porosomes and neosis: some passing thoughts at 70.

This editorial contains some of my reflections on a career spanning almost 50 years in biomedical research at the cellular level and over 12 years as Editor-in-Chief of Cell Biology International, at the time of my 70th birthday. It is gratifying that I have been involved in some of the more important organelles and processes that have come to the forefront of cell research today, and I have chosen just three examples to illustrate this point.

Pegylated derivatives of recombinant human arginase (rhArg1) for sustained in vivo activity in cancer therapy: preparation, characterization and analysis of their pharmacodynamics in vivo and in vitro and action upon hepatocellular carcinoma cell (HCC).

BACKGROUND: Protein used in medicine, e.g. interferon, are immunogenic and quickly broken down by the body. Pegylation is a recognized way of preserving their integrity and reducing immune reactions, and works well with enzymes used to degrade amino acids, a recent focus of attention in controlling cancer growth. Of the two arginine-degrading enzymes being explored clinically, arginine deiminase is a decidedly foreign mycoplasm-derived enzyme, whereas human arginase 1 is a native liver enzyme. Both have been pegylated, the former with adjuncts of 20 kD, the latter with 5 kD PEG. Pegylation is done by several different methods, not all of which are satisfactory or desirable. METHODS: The preparation of novel polyethylene glycol (PEG) derivatives for modifying proteins is described, but directed specifically at pegylation of recombinant human arginase 1 (rhArg1). rhArg1 expressed in Escherichia coli was purified and coupled in various ways with 5 different PEG molecules to compare their protective properties and the residual enzyme activity, using hepatocellular cell lines both in vitro and in vivo. RESULTS: Methoxypolyethylene glycol-succinimidyl propionate (mPEG-SPA 5,000) coupled with very high affinity under mild conditions. The resulting pegylated enzyme (rhArg1-peg5,000 mw) had up to 6 PEG chains of 5K length which not only protected it from degradation and any residual immunogenicity, but most importantly let it retain >90% of its native catalytic activity. It remained efficacious in depleting arginine in rats after a single ip injection of 1,500 U of the conjugate as the native enzyme, plasma arginine falling to >0.05 microM from approximately 170 microM within 20 min and lasting 6 days. The conjugate had almost the same efficacy as unpegylated rhArg1 on 2 cultured human liver cancer (HCC) cell lines. It was considerably more effective than 4 other pegylated conjugates prepared. CONCLUSION: Valuable data on the optimization of the pegylation procedure and choice of ligand that best stabilizes the enzyme arginase 1 are presented, a protocol that should equally fit many other enzymes and proteins. It is a long lasting arginine-depleting enzyme in vivo which will greatly improve its use in anti-cancer therapy.

Rediscovery in science-a second eureka moment? A case in the neovascularization in cancer.

Rediscoveries are not uncommon. However, sometimes they can be more significant than confirmatory or extensions of existing findings, although many authors today refer to them as discoveries in their own right. This has led to papers repeatedly rehearsing the expression "we show here for the first time…". When a finding has opened up a whole new field of research, this is more in line with a true discovery. When particular attention is drawn to such an event by editorials in widely read journals, such as Nature, its importance is bolstered. But if it turns out to be a rediscovery, the implications are considerable and the problem has to be brought to the attention not only of those in the same field of research, but to a wider audience to put the record straight. Consequently, acknowledgment of those who made the original discovery needs to be equally well publicised. A short discussion is presented of ways we might reduce the many claims of "new" discoveries that seem to be of considerable significance but are in fact rediscoveries.

The primary cilium - once a "rudimentary" organelle that is now a ubiquitous sensory cellular structure involved in many pathological disorders.

This article looks mostly at the steps that have led to the primary cilium finding its place in our understanding of cell biology, developmental biology, and medical syndromes due to its aberrations. It is a personal account that stresses, if nothing else, the value of the adage "stick to your guns". My obsession with this organelle, following on from fascination with the centriole, has led to a whole career devoted to determining the nature and role of primary cilia in basic cell biology, which has proved much more important than had been appreciated for almost a century. They are heavily involved in very many aspects of cell physiology that have much wider implications with regard to human biology and probably throughout the animal kingdom. That aberrations, to the surprise of many researchers in their structure or functioning has led to their being implicated or perhaps deeply involved in an extraordinary range of medical conditions. This invitation allows me to raise crucial questions that need answers regarding the regulation of their genesis, their cache of both intracellular and extracellular signal, and their association with a multitude of development processes from embryo to adult status.

Action of Lovastatin (Mevinolin) on an in vitro model of angiogenesis and its co-culture with malignant melanoma cell lines.

BACKGROUND: Lovastatin and other statins may reduce the development of melanomas. The effects on melanoma cells and their ability to enhance angiogenesis in a co-culture system presented an opportunity to assess whether Lovastatin act on melanoma cells, HUVEC or both types of cells. RESULTS: Direct effects of co-culturing two different malignant melanoma cells (A375 and G361) on the process of angiogenesis in vitro was studied with our angiogenesis model, based on human dermal fibroblasts and human umbilical vein endothelial cells (HUVEC). Co-cultures were set up using "sland" and "dispersed seeding" techniques. A statistically significant increase in tubule formation in both cases was observed compared to controls. The effects of doses equivalent to therapeutic concentrations of Lovastatin were analysed. The drug inhibited the growth of all cell types, induced apoptosis, and markedly reduced the formation of tubules in the angiogenesis model at low concentrations. Its action was successfully reversed by the introduction of geranylgeranyl pyrophosphate. CONCLUSION: Lovastatin can reduce both tumour (melanoma) cell growth, and the angiogenic activity of these cells in co-cultures using an established 2-dimensional model angiogenesis system beyond that which would be seen by reduced proliferation alone.

Integrity and stability of the citrulline-arginine pathway in normal and tumour cell lines.

Arginine catabolizing enzymes have been used on cancers for over 60 years. In the last 5 years the ability of arginine catabolizing enzymes, not only to inhibit proliferation, but to kill tumour cells has been reinvestigated. Selectivity of action lies in the inability of many tumours to circumvent arginine deprivation by recycling precursors through the urea cycle. While this offers an immediate window of opportunity to treat, e.g. melanomas and hepatocellular carcinomas (HCC) that have poor citrulline converting ability, it is possible that the deprivation can be applied to many other types of cancer. The problem of deficiency of the urea cycle enzymes in a wider range of normal and malignant cell lines has been addressed, and shown to be variable throughout several different tumour types. We also need to know how fickle recycling enzyme activity can be in both normal and tumour cells, and found to be remarkable stable. Increasing interest is shown in the amino acid (arginine) deprivation protocol because it has already moved into the clinic. Initial findings on a named-patient basis have been encouraging, and the development of a new rational approach to the systemic treatment of melanomas, HCCs and leukemias seems imminent. This is the more attractive because arginine deprivation protocols can also 'stage' tumour cells for combination therapy in cases where they might not be killed outright by deprivation alone.

Cell biology as the basis of a better understanding of cancer.

Clinicians will argue that cancer can only really receive the treatment that is needed through thorough understanding of medicine. However, even empirical approaches to therapy result in experimental analysis of the agencies involved on test cells, usually in culture. From the obverse perspective, cell biologists will argue that until we fully understand cell cycle regulation, tumour management will be too imprecise to make the best advances. A forum is needed whereby the fundamental studies on cells prior to, during and after transformation in vitro can be freely reported (open access) and discussed. The action of anticancer agents and cancer preventative substances can more easily be studied in vitro before the often excessive complexity of making similar studies in experimental and human cancers is tackled. Cancer Cell International is committed to providing such a forum. Ironically within a few months of launching this open access journal, Elsevier had much the same idea, and there one has to pay for the privilege of downloading vital papers in this biomedical field.

Stochastic description of the ligand-receptor interaction of biologically active substances at extremely low doses.

Signalling molecules can be effective at extraordinarily low concentrations (down to attomolar levels). To handle such cases, probabilistic methods have been used to describe the formal kinetics of action of biologically active substances in these low doses, although it has been necessary to review what is meant by such a term. The mean numbers of transformed/degraded molecules and their dispersions were calculated for the possible range of ligand-receptor binding schemes. We used both analytical equations and numerical simulations to calculate the coefficients of variation (ratio of standard deviation to mean) and demonstrated that the distribution of the coefficient is highly dependent on the reaction scheme. It may, therefore, be used as an additional factor for discriminating between cooperative and noncooperative models of ligand-receptor interaction over extreme ranges of ligand dilution. The relevance to signalling behaviour is discussed.

A new journal - "Theoretical Biology and Medical Modelling".

Biology has a conceptual basis that allows one to build models and theorize across many life sciences, including medicine and medically-related disciplines. A dearth of good venues for publication has been perceived during a period when bioinformatics, systems analysis and biomathematics are burgeoning. Steps have been taken to provide the sort of journal with a quick turnaround time for manuscripts which is online and freely accessible to all readers, whatever their persuasion or discipline. We have now been running for some time a journal which has had many good papers presented pre-launch, and a steady stream of papers thereafter. The value of this journal as a new venue has already been vindicated. Within a short space of time, we have founded a state-of-the-art electronic journal freely accessible to all in a much sort-after interdisciplinary field that will be of benefit to the thinking life scientist, which must include medically qualified doctors as well as scientists who prefer to build their new hypotheses on basic principles and sound concepts underpinning biology. At the same time, these principles are not sacrosanct and require critical analysis. The journal http://www.tbiomed.com promises to deliver many exciting ideas in the future.

Possible attenuation of the G2 DNA damage cell cycle checkpoint in HeLa cells by extremely low frequency (ELF) electromagnetic fields.

BACKGROUND: The issue remains unresolved as to whether low frequency magnetic fields can affect cell behaviour, with the possibility that they may be in part responsible for the increased incidence of leukaemia in parts of the population exposed to them. METHODS: Combined treatment of HeLa cells with gamma-irradiation (1, 3 and 5 Grays) and extra low frequency magnetic fields of ~50 Hz was carried out under rigorously controlled conditions. RESULTS: Synchronised cells progressing from S-phase arrived at mitosis on average marginally ahead of irradiation controls not exposed to ELF. In no instance out of a total of twenty separate experiments did this "double-insult" further delay entry of cells into mitosis, as had been anticipated. CONCLUSION: This apparently "non-genotoxic" agent (ELF) appears to be capable of affecting cells that would normally arrest for longer in G2, suggesting a weakening of the stringency of the late cycle (G2) checkpoint.

Recovery of the Decorin-Enriched Fraction, Extract (D), From Human Skin: An Accelerated Protocol.

The original extraction procedure of Engel and Catchpole [1] has often been used to recover decorin-enriched material from the skin. This material has a strong inhibitory effect on fibroblast proliferation, and clearly suppresses it in skin except after the first 5-6 days of wounding when new scaffold material is required. The aim of our present study has been to find and evaluate the product of a faster recovery method, and to check its consistency as a more reliable means of regularly obtaining sufficient material for topical application in wounds that might become hypertrophic. Modifications of the original Toole and Lowther [2] extraction procedure have been carefully evaluated in an attempt to cut preparation time without compromising biological activity of the inhibitory extract. We have devised a faster recovery procedure without compromising biological activity, even if initial recovery has been somewhat reduced. The latter problem could be offset by repeated cycles of the final extraction step. The main inhibitory activity is shown to be within the decorin-enriched "extract D," as the core protein and DSPG II. Adjustment of the extract towards neutrality after dialysis against water keeps most of the extracted protein in solution and yielded a decorin-enriched preparation that had a specific activity equivalent to that of the old method. It also yielded a fraction that was readily lyophilised to give a small amount of material that could be stored indefinitely without loss of activity and readily redissolved in aqueous solution. A reliable and relatively quick method is presented for the production, from human skin, of a decorin-enriched preparation that has strong fibroblast inhibitory action. The value of the procedure is that it is inexpensive and can produce the quantities that might be used topically in reducing hypertrophic scarring of wounds.

Metabolic scaling: consensus or controversy?

BACKGROUND: The relationship between body mass (M) and standard metabolic rate (B) among living organisms remains controversial, though it is widely accepted that in many cases B is approximately proportional to the three-quarters power of M. RESULTS: The biological significance of the straight-line plots obtained over wide ranges of species when B is plotted against log M remains a matter of debate. In this article we review the values ascribed to the gradients of such graphs (typically 0.75, according to the majority view), and we assess various attempts to explain the allometric power-law phenomenon, placing emphasis on the most recent publications. CONCLUSION: Although many of the models that have been advanced have significant attractions, none can be accepted without serious reservations, and the possibility that no one model can fit all cases has to be more seriously entertained.

Arginine catabolism, liver extracts and cancer.

Although it is self evident that cells will not grow in amino acid deficient medium, an observation less well appreciated is that malignant cells are particularly vulnerable to such deprivation, which can lead to their rapid demise. Indeed, the more flagrantly malignant the phenotype (anaplastic the tumor), the more susceptible the cells seem to be to deprivation. While some attempts to employ this strategy in cancer treatment have been made, the difference between normal and malignant cells should be more fully exploited as a means of selectively eliminating tumor cell populations. To be successful, information on differences between the normal and the deranged cell cycle engine and checkpoints, especially how these are affected by deprivation, is of crucial importance. Since it is only recently that the controls at restriction points have been elucidated, it is little surprise that earlier attempts to control tumor cell growth by limiting the availability of an essential amino acid have met with limited success. Studies have been sporadic and isolated, often with little more than anecdotal descriptions as far as clinical work was concerned. This review concentrates on what has been accomplished primarily in vitro and since about 1950 with regard to arginine catabolism, while recognising that other essential amino acids have also been the focus of attention by some investigators. Treatments have included medium and plasma manipulation, dietary control, enzymatic degradation, and the use of liver extracts. On some occasions, substitution of amino acid analogues has been explored. It is argued that current knowledge, combined with past experience, calls for a much closer examination of the full potential of amino acid (and specifically arginine) deprivation as a means of controlling tumor growth, with greater attention to protocols that might be used to treat human cancers.