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Mitochondria have recently emerged as intriguing targets for anticancer drugs. A variety of compounds have been now identified that act via mitochondria. These compounds, termed mitocans (an acronym for mitochondria and cancer), destabilise mitochondria and cause apoptosis, which is, at least in some cases, selective for cancer cells. Mitochondria are the powerhouse of the cell, providing it with energy, as well as the source of important mediators of apoptosis. Recent findings show that individual types of cancers are complex and can differ considerably in their array of DNA mutations, harbouring different sets of genetic causes. This indicates that it will be very unlikely to cure cancer by drugs targeting only a few gene products or single pathways that are essential for tumour survival. What is needed then is an invariant target, common to all cells, but which is predominantly only affected by drugs when delivered inside the cancer cells. Such targets appear to be mitochondria, with very rare mutations, and mitocans can be expected to be very efficient drugs of choice for a number of different types of the neoplastic disease.

Alpha-tocopheryl succinate (alpha-TOS) is a selective inducer of apoptosis in cancer cells, which involves the accumulation of reactive oxygen species (ROS). The molecular target of alpha-TOS has not been identified. Here, we show that alpha-TOS inhibits succinate dehydrogenase (SDH) activity of complex II (CII) by interacting with the proximal and distal ubiquinone (UbQ)-binding site (Q(P) and Q(D), respectively). This is based on biochemical analyses and molecular modelling, revealing similar or stronger interaction energy of alpha-TOS compared to that of UbQ for the Q(P) and Q(D) sites, respectively. CybL-mutant cells with dysfunctional CII failed to accumulate ROS and underwent apoptosis in the presence of alpha-TOS. Similar resistance was observed when CybL was knocked down with siRNA. Reconstitution of functional CII rendered CybL-mutant cells susceptible to alpha-TOS. We propose that alpha-TOS displaces UbQ in CII causing electrons generated by SDH to recombine with molecular oxygen to yield ROS. Our data highlight CII, a known tumour suppressor, as a novel target for cancer therapy.

Arsenic-based compounds have become accepted agents for cancer therapy providing high rates of remission of some cancers such as acute promyelocytic leukemia (APL). The mechanisms by which arsenic-containing compounds kill cells and reasons for selective killing of only certain types of cancer cells such as APLs have recently been delineated. This knowledge was gained in parallel with increasing understanding and awareness of the importance of intracellular redox systems and regulation of the production of reactive oxygen species (ROS) by controlling mitochondrial function. Many of the targets for the arsenic-containing compounds are mitochondrial proteins involved in regulating the production of ROS. Inhibition of these proteins by disulfide linkage of vicinal thiol groups often leads to increased production of ROS and induction of apoptotic signalling pathways. Sensitivity or resistance to the actions of arsenic-containing compounds on cancer cells and normal cells depends on the levels of transport systems for their uptake or efflux from the cells as well as their redox defence mechanisms. The exact mechanisms of arsenic toxicity as well as its anticancer properties are likely to be related and these aspects of arsenic metabolism are covered in this review. Greater understanding of the mechanisms of action of arsenic will help determine the risks of human exposure to this chemical. Novel organic arsenic-containing compounds and the lessons learned from studying their selective sensitivity in targeting dividing endothelial cells to inhibit angiogenesis raise the future possibility for designing better targeted antineoplastic arsenic-containing compounds with less toxicity to normal cells.

"Mitocans" from the vitamin E group of selective anticancer drugs, alpha-tocopheryl succinate (alpha-TOS) and its ether analogue alpha-TEA, triggered apoptosis in proliferating but not arrested endothelial cells. Angiogenic endothelial cells exposed to the vitamin E analogues, unlike their arrested counterparts, readily accumulated reactive oxygen species (ROS) by interfering with the mitochondrial redox chain and activating the intrinsic apoptotic pathway. The vitamin E analogues inhibited angiogenesis in vitro as assessed using the "wound-healing" and "tube-forming" models. Endothelial cells deficient in mitochondrial DNA (mtDNA) were resistant to the vitamin E analogues, both in ROS accumulation and apoptosis induction, maintaining their angiogenic potential. alpha-TOS inhibited angiogenesis in a mouse cancer model, as documented by ultrasound imaging. We conclude that vitamin E analogues selectively kill angiogenic endothelial cells, suppressing tumor growth, which has intriguing clinical implications.

Mitochondria have recently emerged as new and promising targets for cancer prevention and therapy. One of the reasons for this is that mitochondria are instrumental to many types of cell death and often lie downstream from the initial actions of anti-cancer drugs. Unlike the tumour suppressor gene encoding p53 that is notoriously prone to inactivating mutations but whose function is essential for induction of apoptosis by DNA-targeting agents (such as doxorubicin or 5-fluorouracil), mitochondria present targets that are not so compromised by genetic mutation and whose targeting overcomes problems with mutations of upstream targets such as p53. We have recently proposed a novel class of anti-cancer agents, mitocans that exert their anti-cancer activity by destabilising mitochondria, promoting the selective induction of apoptotic death in tumour cells. In this communication, we review recent findings on mitocans and propose a common basis for their mode of action in inducing apoptosis of cancer cells. We use as an example the analogues of vitamin E that are proving to be cancer cell-specific and may soon be developed into efficient anti-cancer drugs.

The search for a selective and efficient anticancer agent for treating all neoplastic disease has yet to deliver a universally suitable compound(s). The majority of established anticancer drugs either are nonselective or lose their efficacy because of the constant mutational changes of malignant cells. Until recently, a largely neglected target for potential anticancer agents was the mitochondrion, showing a considerable promise for future clinical applications. Vitamin E (VE) analogs, epitomized by alpha-tocopheryl succinate, belong to the group of "mitocans" (mitochondrially targeted anticancer drugs). They are selective for malignant cells, cause destabilization of their mitochondria, and suppress cancer in preclinical models. This review focuses on our current understanding of VE analogs in the context of their proapoptotic/anticancer efficacy and suggests that their effect on mitochondria may be amplified by modulation of alternative pathways operating in parallel. We show here that the analogs of VE that cause apoptosis (which translates into their anticancer efficacy) generally do not possess antioxidant (redox) activity and are prototypical of the mitocan group of anticancer compounds. Therefore, by analogy to Oscar Wilde's play The Importance of Being Earnest, we use the motto in the title "the importance of being redox-silent" to emphasize an essentially novel paradigm for cancer therapy, in which redox-silence is a prerequisite property for most of the anticancer activities described in this communication.

Recent evidence suggests that a subset of cells within a tumour have 'stem-like' characteristics. These tumour-initiating cells, distinct from non-malignant stem cells, show low proliferative rates, high self-renewing capacity, propensity to differentiate into actively proliferating tumour cells, resistance to chemotherapy or radiation, and they are often characterised by elevated expression of the stem cell surface marker CD133. Understanding the molecular biology of the CD133(+) cancer cells is now essential for developing more effective cancer treatments. These may include drugs targeting organelles, such as mitochondria or lysosomes, using highly efficient and selective inducers of apoptosis. Alternatively, agents or treatment regimens that enhance sensitivity of these therapy-resistant "tumour stem cells" to the current or emerging anti-tumour drugs would be of interest as well.

Overexpression of erbB2 is associated with resistance to apoptosis. We explored whether high level of erbB2 expression by cancer cells allows their targeting using an erbB2-binding peptide (LTVSPWY) attached to the proapoptotic alpha-tocopheryl succinate (alpha-TOS). Treating erbB2-low or erbB2-high cells with alpha-TOS induced similar levels of apoptosis, whereas alpha-TOS-LTVSPWY induced greater levels of apoptosis in erbB2-high cells. alpha-TOS rapidly accumulated in erbB2-high cells exposed to alpha-TOS-LTVSPWY. The extent of apoptosis induced in erbB2-high cells by alpha-TOS-LTVSPWY was suppressed by erbB2 RNA interference as well as by inhibition of either endocytotic or lysosomal function. alpha-TOS-LTVSPWY reduced erbB2-high breast carcinomas in FVB/N c-neu transgenic mice. We conclude that a conjugate of a peptide targeting alpha-TOS to erbB2-overexpressing cancer cells induces rapid apoptosis and efficiently suppresses erbB2-positive breast tumors.

Recently mitochondria in cancer cells have emerged as the Achilles heel for tumour destruction. Anti-cancer agents specifically targeting cancer cell mitochondria are referred to as 'mitocans'. These compounds act by destabilising these organelles, unleashing their apoptogenic potential, resulting in the efficient death of malignant cells and suppression of tumour growth. Importantly, at least some mitocans are selective for cancer cells, and these are represented by the group of redox-silent vitamin E analogues, epitomised by alpha-tocopheryl succinate (alpha-TOS). This compound has proven itself in pre-clinical models to be an efficient anti-cancer agent, targeting complex II of the respiratory chain to displace ubiquinone binding. We propose that disrupting the electron flow of mitochondrial complex II results in generation of superoxide, triggering mitochondrial destabilisation and initiation of apoptotic pathways. Moreover, alpha-TOS is selective for cancer cells with their reduced anti-oxidant defenses and lower esterase activity than the normal (non-malignant) counterparts. In this mini-review we discuss the emerging significance of mitocans, as exemplified by alpha-TOS.

Mitochondria are proving to be worthy targets for activating specific killing of cancer cells in tumors and a diverse range of mitochondrial targeted drugs are currently in clinical trial to determine their effectiveness as anti-cancer therapies. The mechanism of action of mitochondrial targeted anti-cancer drugs relies on their ability to disrupt the energy producing systems of cancer cell mitochondria, leading to increased reactive oxygen species and activation of the mitochondrial dependent cell death signaling pathways inside cancer cells. We propose that this emerging class of drugs be called "mitocans", a term that reflects their mitochondrial targeting and anti-cancer roles. They are discussed in this review in the context of their mode of action whereby they target the functional differences and altered properties of the mitochondria inside cancerous but not normal cells. Hence, mitocans include drugs affecting the following mitochondrial associated activities: hexokinase inhibitors; electron transport/respiratory chain blockers; activators of the mitochondrial membrane permeability transition pore targeting constituent protein subunits, either the voltage dependent anion-selective channel (VDAC) or adenine nucleotide transporter (ANT); inhibitors of Bcl-2 anti-apoptotic family proteins and Bax/Bid pro-apoptotic mimetics. In particular, a recent surge has occurred in the number of patent documents describing small molecule inhibitors and BH3 mimetic blockers of Bcl-2/Bcl-x(L) function as obvious and important targets for promoting mitochondrial induced cancer cell death and for enhancing the actions of other chemotherapeutic agents. One of the other highly significant results to emerge from clinical applications of mitochondrial targeted drugs as cancer therapies to date is that they have shown limited side-effects on the normal "healthy" cell populations in vivo. It is still too early to judge the clinical impact that mitocans will make in treating cancer. Further clinical studies will be required before these novel drugs become established as single modality anti-cancer therapies or are used in conjunction with existing chemotherapies. However, it is clear from the present studies that mitocans offer great potential as effective and exciting new developments in cancer therapy, providing direct activation of cancer cell death by mitochondrial mediated apoptosis and that this complements the other pathways by which existing treatments kill cancer cells. Undoubtedly, mitocans will become an integral part of modern weaponry in the fight to eliminate cancer.

Mitochondria have emerged recently as effective targets for novel anti-cancer drugs referred to as 'mitocans'. We propose that the molecular mechanism of induction of apoptosis by mitocans, as exemplified by the drug alpha-tocopheryl succinate, involves generation of reactive oxygen species (ROS). ROS then mediate the formation of disufide bridges between cytosolic Bax monomers, resulting in the formation of mitochondrial outer membrane channels. ROS also cause oxidation of cardiolipin, triggering the release of cytochrome c and its translocation via the activated Bax channels. This model may provide a general mechanism for the action of inducers of apoptosis and anticancer drugs, mitocans, targeting mitochondria via ROS production.

Luciferase reporter constructs are widely used for analysis of gene regulation when characterizing promoter and enhancer elements. We report that the recently developed codon-modified Renilla luciferase construct included as an internal standard for cotransfection must be used with great caution with respect to the amount of DNA transfected. Also, the dual-luciferase reporter vectors encoding Photinus pyralis firefly or Renilla reniformis luciferase showed a linear increase in dose-response with increasing amounts of transfected DNA, but at higher levels of transfected DNA, a reduction in expressed levels of luciferase activity resulted. In addition, treatment with type I interferon (IFN) was found to significantly reduce levels of P. pyralis firefly and Renilla luciferase activity. In contrast, cells transfected with a green fluorescent protein (GFP) reporter construct showed no significant IFN-associated change. The reduction in luciferase activity resulting from IFN treatment was not due to IFN-mediated cytotoxicity, as no change in cellular propidium iodide (PI) staining was observed by flow cytometry. IFN treatment did not alter the levels of firefly luciferase activity in cell culture supernatants or the luciferase mRNA levels determined by quantitative real-time RT-PCR analysis. Based on these results, it is probable that the IFN-induced reduction in levels of luciferase activity detected in reporter assays occurs via a posttranscriptional mechanism. Thus, it is important to be aware of these complications when using luciferase reporter systems in general or for analyzing cytokine-mediated responsive regulation of target genes, particularly by the type I IFNs.

Historically, patients with disseminated cancer have had poor prognoses and chemotherapy has been of little benefit. However, several different avenues of clinical research are providing reasons for hope. The advent of cytokine immunotherapy, particularly in combination with chemotherapy (biochemotherapy) has seen significantly improved outcomes for metastatic disease. Early biochemotherapy trials often revealed more than 20% complete responses. Unfortunately, Phase III trials have not confirmed earlier expectations for reasons that are not clear, but may reflect the inclusion of patients with refractory brain or other metastases in later trials. More recently, cancer vaccine therapies have provided significantly improved patient survival rates. It is not uncommon for 5-year survival rates of post-surgical patients recovering from metastatic malignancy who receive cancer vaccine therapy to reach more than 50%. Cytokines have become an integral part of cancer therapy and are also under trial together with cancer vaccines as post-surgical adjuvant therapies providing significant gains in long term survival rates. New insights from several different areas of research into the properties of tumour cells and their significance for immunosurveillance point to the importance of the tumour cells themselves as antigen presenting cells. Recent developments with genetically deficient animals and cancer cells have provided greater understanding at the molecular level of the importance of a functioning antigen presenting system operating inside tumour cells. This new knowledge offers support for further enhancing patient survival by combining previous therapies such as use of cytokines in biochemotherapy together with immunization using cytokine activated whole cell cancer vaccines in the future.

Sequentially treating human melanoma cell lines by priming with interferon-gamma before adding interferon-beta was previously found to be the most efficient protocol for producing concurrently increased expression of the three surface antigens B7-1, intercellular adhesion molecule-1 and human histocompatibility leucocyte antigens Class I. The present study describes similar outcomes when the same sequential intercellular adhesion molecule-based protocol is applied to murine B16-F10 melanoma cells as well as preclinical studies using the B16-F10 model as a poorly immunogenic melanoma. Thus, treating B16-F10 cells or a highly expressing B7-1 transfected subline (B16-F10/B7-1 hi) by priming with interferon-gamma for 24 h before adding interferon-beta for a further 48 h (interferon-gamma 72/beta 48) increased expression of all three surface antigens, particularly major histocompatibility complex class I whose increased expression was sustained for several days. As a whole tumour cell vaccine, interferon-gamma 72/beta 48 treated B16-F10 cells produced greater levels of cytoxic T lymphocyte response compared to vaccines prepared from cells treated with a single type of interferon. Furthermore, B16-F10 cells expressing high levels of B7-1 and treated using the interferon-gamma 72/beta 48 protocol (interferon-gamma 72/beta 48-treated B16-F10/B7-1 hi) produced substantially increased cytoxic T lymphocyte responses with a fivefold greater synergy than the combined results of either interferon treated or B7-1 expressing cells tested individually. The resulting CD8+ cytoxic T lymphocyte showed greater specificity for B16-F10 cells with tenfold higher killing than for syngeneic EL-4 lymphoma cells. Killing proceeded via the perforin-mediated pathway. CTL responses were induced independent of CD4+ T helper cells. The majority of mice receiving interferon-gamma 72/beta 48-treated B16-F10/B7-1 hi vaccine in vivo remained tumour free after challenge with 5 x 105 live B16-F10 cells expressing intermediate B7-1 levels. The novel strategy described will help enhance vaccine potency when applied clinically to prepare whole cell based cancer vaccine therapies.

The transcription factor STAT1 plays a pivotal role in signal transduction of type I and II interferons (IFNs). STAT1 activation leads to changes in expression of key regulatory genes encoding caspases and cell cycle inhibitors. Deficient STAT1 expression in human cancer cells and virally mediated inhibition of STAT1 function have been associated with cellular resistance to IFNs and mycobacterial infection in humans. Thus, given the relative importance of STAT1, we isolated and characterized a human STAT1 intronic enhancer region displaying IFN-regulated activity. Functional analyses by transient expression identified a repressor region and type I and II IFN-inducible elements within the STAT1 enhancer sequence. A candidate IRF-E/GAS/IRF-E (IGI) sequence containing GAAANN nucleotide repeats was shown by gel shift assay to bind to IFN regulatory factor-1 (IRF-1), but not to IFN-stimulated gene factor-3 (ISGF-3) or STAT1-3. An additional larger IGI-binding complex containing IRF-1 was identified. Mutation of the GAAANN repeats within the IGI DNA element eliminated IRF-1 binding and the IFN-regulated activity of the STAT1 intronic enhancer region. Transfection of the IFN-resistant MM96 cell line to express increased levels of IRF-1 protein also elevated STAT1, STAT2, and p48/IRF-9 expression and enhanced cellular responsiveness to IFN-beta. Reciprocating regulation between IRF-1 and STAT1 genes and encoded proteins indicates that an intracellular amplifier circuit exists controlling cellular responsiveness to the IFNs.

We report the cloning and sequencing of a full-length cDNA encoding a new member of the human IFI54 (HGMW-approved symbol IFIT2) gene family, designated IFI60 (HGMW-approved symbol IFIT4). The upstream regulatory region of IFI60 shows conservation in structure with that of the IFI54 and IFI56 (HGMW-approved symbol IFIT1) genes, each containing two interferon-stimulated response elements upstream of a conserved TATA box. We have established a partial gene map of the IFI54 gene family by analysis of YAC library clones. All four members of the human family are clustered together at chromosome 10q23.3. It is proposed that the four members of the IFI54 gene family evolved by a series of duplication events from a common gene of origin., (Copyright 1998 Academic Press.)

IFN-stimulated gene factor 3 (ISGF3) mediates transcriptional activation of IFN-sensitive genes (ISGs). The component subunits of ISGF3, STAT1alphabeta, STAT2, and p48-ISGF3gamma, are tyrosine phosphorylated before their assembly into a complex. Subsequently, the ISGF3 complex is translocated to the nucleus. We have recently established that the responsiveness of human melanoma cell lines to type I IFNs correlates directly with their intracellular levels of ISGF3 components, particularly STAT1. In the present study, we show that pretreating IFN-resistant melanoma cell lines with IFN-gamma (IFN-gamma priming) before stimulation with type I IFN also results in increased levels of ISGF3 components and enhanced DNA-binding activation of ISGF3. In addition, IFN-gamma priming of IFN-resistant melanoma cell lines increased expression of type I IFN-induced ISG products, including ISG54, 2'-5'-oligoadenylate synthase, HLA class I, B7-1, and ICAM-1 Ags. Furthermore, IFN-gamma priming enhanced the antiviral effect of IFN-beta on the IFN-resistant melanoma cell line, MM96. These results support a role for IFN-gamma priming in up-regulating ISGF3, thereby augmenting the responsiveness of IFN-resistant melanoma cell lines to type I IFN and providing a molecular basis and justification for using sequential IFN therapy, as proposed by others, to enhance the use of IFNs in the treatment of melanoma.

The mechanism of IFN resistance was examined in three long-term cell lines, SK-MEL-28, SK-MEL-3, and MM96, exhibiting significant variation in responsiveness to the antiproliferative and antiviral effects of type I IFNs. The JAK-STAT components involved in IFN signal transduction were analyzed in detail. After exposure to IFN, activation of the IFN type I receptor-linked tyrosine kinases, JAK-1 and TYK-2, was detected at similar levels in both IFN-sensitive and IFN-resistant cell types, indicating that IFN resistance did not result from a deficiency in signaling at the level of receptor-associated kinase activation. However, analysis of ISGF3 transcription factor components, STAT1, STAT2, and p48-ISGF3gamma, revealed that their expression and activation correlated with cellular IFN responsiveness. The analysis was extended to also include IFN-sensitive primary melanocytes, three additional IFN-resistant melanoma cell lines, and seven cell cultures recently established from melanoma patient biopsies. It was consistently observed that the most marked difference in ISGF3 was a lack of STAT1 in the resistant versus the sensitive cells. Transfection of the IFN-resistant MM96 cell line to express increased levels of STAT1 protein partially restored IFN responsiveness in an antiviral assay. We conclude that a defect in the level of STAT1 and possibly all three ISGF3 components in IFN-resistant human melanoma cells may be a general phenomenon responsible for reduced cellular responsiveness of melanomas to IFNs.

Clinical and experimental studies examining the action of IFNs on human malignant melanomas and melanoma cell lines have shown that this cancer cell type is frequently IFN resistant. In the present study, the IFN responsiveness of five melanoma cell lines, SK-MEL-28, SK-MEL-3, MM96, HT-144, and Hs 294T, as determined by the levels of IFN-induced expression of the antiviral proteins, 100 kDa 2',5'-oligoadenylate synthetase (OAS) and Mx Ag, was shown to correlate with the IFN responsiveness of the five lines measured in antiproliferative and antiviral assays. Three of the lines, SK-MEL-28 (IFN sensitive), SK-MEL-3 (moderately IFN sensitive), and MM96 (IFN insensitive) were analyzed further to ascertain their relative levels of IFN-activated signal transduction. Pretreatment of the three melanoma cell lines with the tyrosine kinase inhibitors, Herbimycin A or Genistein, produced a dose-dependent inhibition of the antiviral action of IFN-alpha, -beta, and -gamma and the induction of OAS by IFN-beta. Thus, induction of the antiviral state in melanoma cells by IFN requires activation of tyrosine kinase-dependent signaling pathways. Furthermore, the IFN responsiveness of three melanoma cell lines could be correlated with the ability to detect by immunoblotting of SDS-PAGE displays of cell lysates, IFN-induced tyrosine phosphorylated cellular proteins in the range m.w. 80 to 130 kDa. This induction was also sensitive to the tyrosine kinase inhibitors Herbimycin A and Genistein. Based on these results, we propose that the IFN-resistant melanoma cell lines examined contain a deficiency early in the IFN signal transduction pathway resulting in a reduced potential for IFN-induced tyrosine phosphorylation and a lack of responsiveness to IFN.

Melanoma cell lines exhibit strikingly different sensitivity to the antiproliferative effects of interferon. cDNAs encoding the Type I interferon receptor subunit were amplified by polymerase chain reaction, using as template RNA isolated from three melanoma cell lines displaying greater than 100 fold range in their sensitivity to the antiproliferative effects of IFN-beta. Comparison of the cDNA sequences obtained with the published cDNA sequence from the highly interferon-sensitive lymphoid cell line Daudi revealed only one base change that leads to a conservative amino acid substitution. It is concluded that the cellular differences in responsiveness to interferon, of the melanoma cell lines tested, do not arise from the expression of variants of the cloned Type I interferon receptor subunit.

The design of artificial heart valves has traditionally been based on the development of a prototype device which was then subjected to extensive laboratory testing in order to confirm its suitability for clinical use. In the past the in vitro assessment of a valve's performance was based principally on the measurement of parameters such as pressure difference, regurgitation and, more recently, energy losses. Such measurements can be defined as being at the 'macro' level and rarely show any clinically significant differences amongst currently available prostheses. The analytical approach to flow through heart valves has previously been hampered by difficulties experienced in solving the relevant equations of flow particularly in the case of pulsatile conditions. Computational techniques are now available which enable appropriate solutions to be obtained for these problems and consequently provide an opportunity for detailed examination of the 'micro' level of flow disturbances exhibited by the different valves. This present preliminary study is designed to illustrate the use of such an analytical approach to the flow through prosthetic valves. A single topic has been selected for this purpose which is the comparative value of steady versus pulsatile flow testing. A bileaflet valve was chosen for the analysis and a mathematical model of this valve in the aortic position of the Sheffield Pulse Duplicator was created. The theoretical analysis was carried out using a commercially available Computational Fluid Dynamics package, namely, FIDAP, on a SUN MICROSYSTEMS 10-30 workstation.(ABSTRACT TRUNCATED AT 250 WORDS)

In this paper the specific mitochondrial respiratory chain inhibitors rotenone and antimycin A and the highly specific mitochondrial ATP-synthase inhibitor oligomycin are shown to induce an apoptotic suicide response in cultured human lymphoblastoid and other mammalian cells within 12-18 h. The mitochondrial inhibitors do not induce apoptosis in cells depleted of mitochondrial DNA and thus lacking an intact mitochondrial respiratory chain. Apoptosis induced by respiratory chain inhibitors is not inhibited by the presence of Bcl-2. We discuss the possible role of mitochondrial induced apoptosis in the ageing process and age-associated diseases.

Colourimetric assays which rely on the conversion of metabolic dyes are becoming increasingly used for measuring cell proliferation and cytotoxicity. We developed a method for measuring cellular responsiveness to interferons based on the property of interferons to induce cell defenses to virus-mediated killing. The assay has several advantages over previous assays for measuring anti-viral activity and efficiently detected cytoprotective responses to human interferon of five human melanoma cell lines infected with Semliki Forest virus. The melanoma cell lines showed a varying cytoprotective response to interferons alpha 2, alpha 4, beta and gamma, in agreement with the results of previous assays for measuring melanoma cell responsiveness to interferons based upon the inhibition of cell growth (1).

The protein-tyrosine kinases (PTKs) are a burgeoning family of proteins, each of which bears a conserved domain of 250 to 300 amino acids capable of phosphorylating substrate proteins on tyrosine residues. We recently exploited the existence of two highly conserved sequence elements within the catalytic domain to generate PTK-specific degenerate oligonucleotide primers (A. F. Wilks, Proc. Natl. Acad. Sci. USA 86:1603-1607, 1989). By application of the polymerase chain reaction, portions of the catalytic domains of several novel PTKs were amplified. We describe here the primary sequence of one of these new PTKs, JAK1 (from Janus kinase), a member of a new class of PTK characterized by the presence of a second phosphotransferase-related domain immediately N terminal to the PTK domain. The second phosphotransferase domain bears all the hallmarks of a protein kinase, although its structure differs significantly from that of the PTK and threonine/serine kinase family members. A second member of this family (JAK2) has been partially characterized and exhibits a similar array of kinase-related domains. JAK1 is a large, widely expressed membrane-associated phosphoprotein of approximately 130,000 Da. The PTK activity of JAK1 has been located in the C-terminal PTK-like domain. The role of the second kinaselike domain is unknown.

Rupture of the oesophagus occurred during the application of cricoid pressure at induction of anaesthesia when the patient vomited. The patient, who was bleeding from a gastric ulcer, was found to have a lower oesophageal tear which, although repaired at operation, resulted in a fatal mediastinitis.

1. Rats were fed on diets enriched with starch, sucrose, corn oil or beef tallow for 3 weeks and the activities of various enzymes in the liver were measured. 2. The mitochondrial glycerol phosphate acyltransferase activity was lower in rats fed on the starch diet than on the two high-fat diets. 3. The non-microsomal (presumably peroxisomal) dihydroxyacetone phosphate acyltransferase activity was higher in rats fed on the starch diet and corn-oil diets than in those fed on the sucrose and beef-tallow diets. Urate oxidase activity was higher in rats fed on the starch diet than in the three other groups. There were no significant differences in the activity of acyl-CoA oxidase among the groups. 4. The activity of soluble phosphatidate phosphohydrolase was not significantly different among the dietary groups. There were increases of 3.3--4.3-fold in this activity in the dietary groups 6h after injection of corticotropin. The equivalent increases for the mitochondrial glycerol phosphate acyltransferase were 1.4--1.6 fold. 5. The corticosterone responses to the corticotropin injection were not significantly different between dietary groups. However, the corticosterone response of the rats fed on the two high-fat diets was prolonged when the rats were given an acute load of fructose [Brindley, Cooling, Glenny, Burditt & McKechnie (1981) Biochem. J. 200. 275--283]. 6. Rats fed on the high-fat diets had higher concentrations of circulating cholesterol than those fed on the starch and sucrose diets. Serum triacylglycerol concentrations were lower in the rats fed on the starch diet than in the three other groups. 7. The results are discussed in terms of the relationship between diet, hormonal balance and hepatic glycerolipid metabolism.

A panel of monoclonal antibodies that bind to the murine pluripotential stem cell CFU-s was used to examine the antigenic profile of the stem cell during ontogeny. The results show that the stem cell surface changes dramatically during development. One group of three independently derived monoclonal antibodies binds to subpopulations (50%-70%) of stem cells at plateau values, and these populations increase marginally during development. A second group of four monoclonal antibodies, including anti-H-2Kk (11-4.1), define stem cell antigens that increase from low levels in the fetal liver to high levels in adult bone marrow. The presence of these two classes of antigens on adult splenic stem cells was in general similar to that observed on adult bone marrow. Antigens defined by the first group of monoclonal antibodies were present in similar amounts on CBA, C57B1/6, and Balb/c bone marrow stem cells, whereas antigens of the second group showed mouse strain variations. Quantitative absorption analysis was used to distinguish H-2Kk (11-4.1) from 9F6, which showed a similar developmental profile. Monoclonal antibodies recognizing subpopulations of stem cells were shown to be distinct by complementation studies and recognized antigens not present on brain tissue.

Clonogenic tumor cells and normal stem cells share the property of extensive proliferative potential. Normal stem cells are under stringent growth restraint and respond to appropriate differentiation signals, whereas tumor stem cells have lost the ability to respond normally to these controls. In an attempt to define cell surface molecules involved in the control of hemopoietic cell proliferation and differentiation, we have produced 5 monoclonal antibodies against antigens held in common between hemopoietic stem cells and the Abelson virus-induced pre-B-lymphoma cells from which they were derived. Four of these monoclonal antibodies produced greater than 90% reduction of spleen colony-forming cells, whereas the other bound to a subpopulation (60 to 70%) of spleen colony-forming cells at plateau values. The expression of antigens recognized by these and two other anti-stem cell monoclonal antibodies has been shown to correlate with the differentiation status of a panel of tumor cell lines, with greater expression being observed on cells more closely resembling the pluripotent stem cell than mature hemopoietic cells. Immunoperoxidase staining of bone marrow showed that these antigens are mainly expressed by monocytes and blast cells. Treatment of bone marrow cells with those antibodies which extensively inhibited spleen colony formation and with rabbit complement abolished the ability of progenitor cells to form colonies in soft agar. Quantitative absorption studies distinguished the antigens recognized by two of the anti-stem cell monoclonal antibodies from those detected by anti-H-2k 11-4.1 monoclonal antibody. These observations suggest that the antigens involved may play a role in the regulation of growth and differentiation of stem cells and undifferentiated leukemic cells.

Hybridomas secreting monoclonal antibodies cross-reacting with the mouse bone marrow stem cell (CFUs) have been derived from rats immunized with mouse thymocytes and platelets, cells which have been shown to exhibit antigenic lineage relationships with the stem cell. Anti-stem cell activity in hybridoma supernatants occurs at low frequency. Of 418 hybridomas screened, 6 showed initial positivity while two yielded stable cloned cell lines. Both of these monoclonal antibodies, 1C10 and 13C6, bind to subpopulations of stem cells in the bone marrow resulting in 50-65% inhibition of spleen colony formation in lethally irradiated mice. Protein-A binding studies showed the antibodies to be rat IgG. Quantitative absorption and indirect binding studies suggest that the antigens recognized by 1C10 and 13C6 monoclonal antibodies are minor components of the inoculating cell membrane. 1C10 and 13C6 bind to minor subpopulations of cells in the bone marrow, 13 and 5% of cells being rosetted, respectively, at plateau values. These results indicate that, used in conjunction with other monoclonal antibodies or selection tecniques, these antibodies may be useful in stem cell purification.

Structural variation in the primary structure of human T200 glycoprotein has been detected. Three cDNA variants have been characterized each of which encode T200 molecules that differ in size as a result of sequence differences in their amino-terminal regions. The largest form of the molecule is distinguished from the smallest by an insert of 161 amino acids, after the first eight amino-terminal residues. The other variant has an insert at the same location of 47 amino acids identical to residues 75-121 in the larger insert. Both extra domains are rich in serine and threonine residues and are likely to display multiple O-linked oligosaccharides. These structural variants which probably arise by cell-type-specific alternative splicing provide a molecular basis for the previously observed structural and antigenic heterogeneity of T200 glycoprotein. In addition to the variable amino-terminal region, the external domain of human T200 glycoprotein consists of a second cysteine-rich region of about 400 amino acids, a single transmembrane-spanning region and a large cytoplasmic domain of 707 amino acids shared by all of the structural variants and highly conserved between species. The gene encoding human T200 is located on the long arm of chromosome 1.

Cross-linking reovirus spike protein with the bifunctional reagent dimethyl suberimidate revealed that each spike was composed of a pentameric aggregate of polypeptide lambda 2.

A panel of monoclonal antibodies has been used to investigate the presence of antigens of the 'T200' family of cell surface glycoproteins on hemopoietic stem cells. As measured by spleen colony inhibition using CBA bone marrow, stem cells were shown to be antigenically heterogeneous with respect to 'T200' family antigens; greater than 60% loss of spleen colonies was observed with 74/8 and 30F11 monoclonal antibodies and with Ly5.1 alloantiserum, whereas little effect was observed with 30G12. Two of four monoclonal antibodies with specificity for B220 showed significant inhibition of spleen colony formation. Monoclonal antibodies against Mac-1, another family of cell surface glycoproteins, had little effect on stem cells. In general, a similar pattern of spleen colony inhibition was observed in CBA, BALB/c, and C57/BL/6 mice. Spleen colony inhibition did not correlate with antibody isotype or with antibody binding to thymocytes, B lymphocytes, macrophages, or platelets as determined by indirect radioimmunoassay. Cell lineage antigens held in common between thymocytes and stem cells were shown to be wholly represented on the T200 glycoprotein. Thus, the anti-stem cell activity in anti-thymocyte serum (ATS) was absorbed by BW5147 T200+ cells but not by a T200- variant. The results show that some antigens of the 'T200' family of cell surface glycoproteins defined with monoclonal antibodies are expressed on stem cells and that other T200 antigens represented in ATS but not as yet identified with monoclonal antibodies show T lineage restriction.

Degenerate oligodeoxyribonucleotide (oligo) primers derived from amino acid (aa) sequence motifs held in common between all members of the protein tyrosine kinase (PTK) family were used to prime the amplification of PTK-related sequences from a variety of murine cDNA sources, including the haemopoietic cell lines, FDC-P1 and WEHI-3B D+, peritoneal macrophages and whole brain. Several parameters, such as the length (short, i.e., less than 20 nucleotides (nt) vs. long, i.e., greater than 30 nt) and degeneracy (i.e., moderately degenerate vs. highly degenerate) of the oligo primers and the temperature of the extension phase of the reaction, were examined. The data from these analyses suggest that the most effective type of primer in this application of the polymerase chain reaction is a short, moderately degenerate oligo such as that which might be derived from the small patches of aa sequence homology that are frequently found to be held in common among members of protein families. In addition to a number of previously described PTK sequences, a novel mammalian PTK-related sequence was uncovered.