• Keratinocyte growth factor increases hair follicle survival following cytotoxic insult.

      Booth, Catherine; Potten, Christopher S; CRC Epithelial Biology Group, Paterson Institute, Christie Hospital (NHS) Trust, Manchester, U.K. cbooth@picr.man.ac.uk (2000-04)
      Hair loss is a distressing side-effect of cancer therapy. Factors that might reduce this loss are therefore likely to improve patient well-being and reduce treatment refusal. Keratinocyte growth factor has been shown to regulate proliferation and differentiation in epithelial tissues and may regulate the clonogenic cells (stem cells) of the hair follicle. Using X-irradiation as a model cytotoxic agent we investigated whether keratinocyte growth factor pretreatment could increase hair follicle survival (by implication clonogen survival) and regeneration of differentiated progeny (a hair). Irradiated telogen follicle survival data were consistent with that published previously. Daily keratinocyte growth factor pretreatment increased hair survival during the first hair growth cycle, the level of protection having a slight radiation dose dependence. Protection was maintained after a second hair cycle, but at a lower level (hairs and follicles). Hairs irradiated in anagen and analyzed during the second cycle exhibited a similar level of protection. No difference in protection levels could be observed between mice treated either once or twice daily with keratinocyte growth factor. Results indicated approximately 10 extra hairs per mm2 (14.5% of unirradiated control) could survive the cytotoxic insult if pretreated with 12.5 microg keratinocyte growth factor. This could be doubled by a 10 x higher keratinocyte growth factor dose. The fact that protection was maintained during two cycles of hair growth indicated that the clonogenic cells had been protected. Keratinocyte growth factor pretreatment had no significant effect on the level of mitosis but increased the number of p21waf1 expressing cells. Intriguingly, the number of apoptotic fragments per follicle were transiently increased in the keratinocyte growth factor pretreated mice, although this effect was most pronounced in the upper half of the follicle bulb, i.e., above (rather than within) the germinal matrix.
    • Keratinocyte growth factor protects mice from chemotherapy and radiation-induced gastrointestinal injury and mortality.

      Farrell, C L; Bready, J V; Rex, K L; Chen, J N; DiPalma, C R; Whitcomb, K L; Yin, S; Hill, D C; Wiemann, B; Starnes, C O; et al. (1998-03-01)
      Keratinocyte growth factor (KGF) stimulates the proliferation and differentiation of epithelial cells including those of the gastrointestinal tract. Although chemotherapeutics and radiation exposure kill rapidly proliferating tumor cells, rapidly dividing normal cells of the host's gastrointestinal tract are also frequently damaged, leading to the clinical condition broadly termed "mucositis." In this report, recombinant human KGF used as a pretreatment in several mouse models of chemotherapy and/or radiation-induced gastrointestinal injury significantly improved mouse survival. Using multiple-dose 5-fluorouracil, methotrexate, and radiation in combination and total body radiation alone models, KGF increased survival by 55% or greater. In the models that used chemotherapy with or without radiation, KGF significantly ameliorated weight loss after injury and accelerated weight gain during recovery. The basis of these systemic benefits appears to be due in part to the trophic effects of the growth factor on the intestinal epithelium because KGF pretreatment caused an increase in measures of mucosal thickness (villus height and crypt depth) that persisted during the course of 5-fluorouracil chemotherapy. Treatment with KGF also afforded a 3.5-fold improvement in crypt survival in the small intestine, suggesting that KGF also has a direct effect on the crypt stem cells. These data indicate that KGF may be therapeutically useful to lessen the intestinal side effects of current cancer therapy regimens.
    • Keratinocyte proliferative hierarchies confer protective mechanisms in surface epithelia.

      Hume, W J; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester M20 9BX, England. (1985-04)
    • Keratinocyte stem cells: a commentary.

      Potten, Christopher S; Booth, Catherine; Epithelial Biology Department, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, U.K. c.potten@epistem.co.uk (2002-10)
      For many years it has been widely accepted that stem cells play a crucial role in adult tissue maintenance. The concept that the renewing tissues of the body contain a small subcompartment of self-maintaining stem cells, upon which the entire tissue is dependent, is also now accepted as applicable to all renewing tissues. Gene therapy and tissue engineering are driving considerable interest in the clinical application of such hierarchically organized cellular compartments. Recent initial observations have provided a tantalizing insight into the large pluripotency of these cells. Indeed, scientists are now beginning to talk about the possible totipotency of some adult tissue stem cells. Such work is currently phenomenologic, but analysis of data derived from genomics and proteomics, identifying the crucial control signals involved, will soon provide a further impetus to stem cell biology with far reaching applications. The epidermis with its relatively simple structure, ease of accessibility, and the ability to grow its cells in vitro is one obvious target tissue for testing stem cell manipulation theories. It is crucial, however, that the normal keratinocyte stem cell is thoroughly characterized prior to attempting to manipulate its pluripotency. This commentary assesses the data generated to date and critically discusses the conclusions that have been drawn. Our current level of understanding, or lack of understanding, of the keratinocyte stem cell is reviewed.
    • Ketones and lactate "fuel" tumor growth and metastasis: Evidence that epithelial cancer cells use oxidative mitochondrial metabolism.

      Bonuccelli, G; Tsirigos, A; Whitaker-Menezes, D; Pavlides, S; Pestell, R G; Chiavarina, B; Frank, P G; Flomenberg, N; Howell, Anthony; Martinez-Outschoorn, U E; et al. (2010-09)
      Previously, we proposed a new model for understanding the "Warburg effect" in tumor metabolism. In this scheme, cancer-associated fibroblasts undergo aerobic glycolysis and the resulting energy-rich metabolites are then transferred to epithelial cancer cells, where they enter the TCA cycle, resulting in high ATP production via oxidative phosphorylation. We have termed this new paradigm "The Reverse Warburg Effect." Here, we directly evaluate whether the end-products of aerobic glycolysis (3-hydroxy-butyrate and L-lactate) can stimulate tumor growth and metastasis, using MDA-MB-231 breast cancer xenografts as a model system. More specifically, we show that administration of 3-hydroxy-butyrate (a ketone body) increases tumor growth by ∼2.5-fold, without any measurable increases in tumor vascularization/angiogenesis. Both 3-hydroxy-butyrate and L-lactate functioned as chemo-attractants, stimulating the migration of epithelial cancer cells. Although L-lactate did not increase primary tumor growth, it stimulated the formation of lung metastases by ∼10-fold. Thus, we conclude that ketones and lactate fuel tumor growth and metastasis, providing functional evidence to support the "Reverse Warburg Effect". Moreover, we discuss the possibility that it may be unwise to use lactate-containing i.v. solutions (such as Lactated Ringer's or Hartmann's solution) in cancer patients, given the dramatic metastasis-promoting properties of L-lactate. Also, we provide evidence for the up-regulation of oxidative mitochondrial metabolism and the TCA cycle in human breast cancer cells in vivo, via an informatics analysis of the existing raw transcriptional profiles of epithelial breast cancer cells and adjacent stromal cells. Lastly, our findings may explain why diabetic patients have an increased incidence of cancer, due to increased ketone production, and a tendency towards autophagy/mitophagy in their adipose tissue.
    • Ketones and lactate increase cancer cell "stemness," driving recurrence, metastasis and poor clinical outcome in breast cancer: achieving personalized medicine via Metabolo-Genomics.

      Martinez-Outschoorn, U E; Prisco, M; Ertel, A; Tsirigos, A; Lin, Z; Pavlides, S; Wang, C; Flomenberg, N; Knudsen, E S; Howell, Anthony; et al. (2011-04-15)
      Previously, we showed that high-energy metabolites (lactate and ketones) "fuel" tumor growth and experimental metastasis in an in vivo xenograft model, most likely by driving oxidative mitochondrial metabolism in breast cancer cells. To mechanistically understand how these metabolites affect tumor cell behavior, here we used genome-wide transcriptional profiling. Briefly, human breast cancer cells (MCF7) were cultured with lactate or ketones, and then subjected to transcriptional analysis (exon-array). Interestingly, our results show that treatment with these high-energy metabolites increases the transcriptional expression of gene profiles normally associated with "stemness," including genes upregulated in embryonic stem (ES) cells. Similarly, we observe that lactate and ketones promote the growth of bonafide ES cells, providing functional validation. The lactate- and ketone-induced "gene signatures" were able to predict poor clinical outcome (including recurrence and metastasis) in a cohort of human breast cancer patients. Taken together, our results are consistent with the idea that lactate and ketone utilization in cancer cells promotes the "cancer stem cell" phenotype, resulting in significant decreases in patient survival. One possible mechanism by which these high-energy metabolites might induce stemness is by increasing the pool of Acetyl-CoA, leading to increased histone acetylation, and elevated gene expression. Thus, our results mechanistically imply that clinical outcome in breast cancer could simply be determined by epigenetics and energy metabolism, rather than by the accumulation of specific "classical" gene mutations. We also suggest that high-risk cancer patients (identified by the lactate/ketone gene signatures) could be treated with new therapeutics that target oxidative mitochondrial metabolism, such as the anti-oxidant and "mitochondrial poison" metformin. Finally, we propose that this new approach to personalized cancer medicine be termed "Metabolo-Genomics," which incorporates features of both 1) cell metabolism and 2) gene transcriptional profiling. Importantly, this powerful new approach directly links cancer cell metabolism with clinical outcome, and new therapeutic strategies for inhibiting the TCA cycle and mitochondrial oxidative phosphorylation in cancer cells.
    • A key role for Ctf4 in coupling the MCM2-7 helicase to DNA polymerase alpha within the eukaryotic replisome.

      Gambus, Agnieszka; Van Deursen, Frederick; Polychronopoulos, Dimitrios; Foltman, Magdalena; Jones, Richard C; Edmondson, Ricky D; Calzada, Arturo; Labib, Karim; Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester, Manchester, UK. (2009-10-07)
      The eukaryotic replisome is a crucial determinant of genome stability, but its structure is still poorly understood. We found previously that many regulatory proteins assemble around the MCM2-7 helicase at yeast replication forks to form the replisome progression complex (RPC), which might link MCM2-7 to other replisome components. Here, we show that the RPC associates with DNA polymerase alpha that primes each Okazaki fragment during lagging strand synthesis. Our data indicate that a complex of the GINS and Ctf4 components of the RPC is crucial to couple MCM2-7 to DNA polymerase alpha. Others have found recently that the Mrc1 subunit of RPCs binds DNA polymerase epsilon, which synthesises the leading strand at DNA replication forks. We show that cells lacking both Ctf4 and Mrc1 experience chronic activation of the DNA damage checkpoint during chromosome replication and do not complete the cell cycle. These findings indicate that coupling MCM2-7 to replicative polymerases is an important feature of the regulation of chromosome replication in eukaryotes, and highlight a key role for Ctf4 in this process.
    • A key role for the GINS complex at DNA replication forks

      Labib, Karim; Gambus, Agnieszka; Cancer Research U.K., Paterson Institute for Cancer Research, University of Manchester, Manchester, UK. klabib@picr.man.ac.uk <klabib@picr.man.ac.uk> (2007-06)
      The GINS complex is the most recently identified component of the eukaryotic DNA replication machinery and is required both for the initiation of chromosome replication and also for the normal progression of DNA replication forks. Several recent studies suggest that GINS associates at replication forks with the MCM helicase that is responsible for unwinding the parental DNA duplex. Archaea also have an equivalent GINS complex that can interact with MCM and other replisome components. It seems likely that GINS couples MCM to other key proteins at forks, and we discuss here the current literature regarding this important late-comer to the DNA replication field.
    • Key steps in vaccine development

      Stern, Peter L; University of Manchester, UK (2020)
      OBJECTIVE: The goal of a vaccine is to prime the immune response so the immune memory can facilitate a rapid response to adequately control the pathogen on natural infection and prevent disease manifestation. This article reviews the main elements that provide for the development of safe and effective vaccines Data Sources: Literature covering target pathogen epidemiology, the key aspects of the functioning immune response underwriting target antigen selection, optimal vaccine formulation, preclinical and clinical trial studies necessary to deliver safe and efficacious immunization. STUDY SELECTIONS: Whole live, inactivated, attenuated or partial fractionated organism based vaccines are discussed in respect of the balance of reactogenicity and immunogenicity. The use of adjuvants to compensate for reduced immunogenicity is described. The requirements from preclinical studies, including establishing a proof of principle in animal models, the design of clinical trials with healthy volunteers that that lead to licensure and beyond are reviewed. RESULTS: The three vaccine development phases, preclinical, clinical and post licensure integrate the requirements to ensure safety, immunogenicity and efficacy in the final licensed product. Continuing monitoring of efficacy and safety in the immunized populations is essential to sustain confidence in vaccination programs. CONCLUSION: In an era of increasing vaccine hesitancy the need for a better and widespread understanding of how immunization acts to counteract the continuing and changing risks from the pathogenic world is required. This demands a societal responsibility for obligate education on the benefits of vaccination, which as a medical intervention has saved more lives than any other procedure.
    • KEYNOTE-966 trial in progress: Pembrolizumab plus gemcitabine and cisplatin for advanced biliary tract cancer

      Valle, Juan W; Kelley, R. K.; Furuse, J.; Edeline, J.; Finn, R. S.; Ren, Z.; Su, S. C.; Malhotra, U.; Siegel, A. B.; Vogel, A.; et al. (2020)
      Background: Biliary tract cancer (BTC), comprising intra- and extra-hepatic cholangiocarcinoma and gallbladder cancer, is a rare and aggressive malignancy. Most patients (pts) present with advanced or unresectable disease, for which the current standard of care is gemcitabine plus cisplatin. Median survival for these pts is only 12 months, highlighting the need for more effective therapies. Pembrolizumab is a PD-1inhibitor that has demonstrated modest antitumor activity as monotherapy in pts with previously treated BTC and has improved survival when used in combination with platinum-based chemotherapy in other cancer types.
    • Kin4 kinase delays mitotic exit in response to spindle alignment defects.

      Pereira, Gislene; Schiebel, Elmar; Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK. gislene.pereira@manchester.ac.uk (2005-07-22)
      For many polarized cells, it is critical that the mitotic spindle becomes positioned relative to the polarity axis. This is especially important in yeast, where the site of cytokinesis is predetermined. The spindle position checkpoint (SPOC) therefore delays mitotic exit of cells with a mispositioned spindle. One component of the SPOC is the Bub2-Bfa1 complex, an inhibitor of the mitotic exit network (MEN). Here, we show that the Kin4 kinase is a component of the SPOC and as such is essential to delay cell cycle progression of cells with a misaligned spindle. When spindles are correctly oriented, Kin4 and Bub2-Bfa1 are asymmetrically localized to opposite spindle pole bodies (SPBs). Bub2-Bfa1 then becomes inhibited by Cdc5 polo kinase with anaphase onset, a prerequisite for mitotic exit. In response to spindle misalignment, Kin4 and Bub2-Bfa1 are brought together at both SPBs. Kin4 now maintains Bub2-Bfa1 activity by counteracting Cdc5, thereby inhibiting mitotic exit.
    • Kinetic response of haemopoietic cell lineages to growth factors in vivo: their relationship to the microarchitecture of the tissue and its microenvironment.

      Lord, Brian I; Department of Experimental Haematology, Paterson Institute for Cancer Research, Christie Hospital and Holt Radium Institute, Manchester, United Kingdom. (1990)
    • Kinetics and mechanism of the reduction of ferricytochrome c by the superoxide anion.

      Butler, John; Koppenol, W H; Margoliash, E; Department of Biophysical Chemistry, Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester, M20 9BX, United Kingdom (1982-09-25)
      The temperature and pH dependence of the reaction of the superoxide radical anion with ferricytochrome c have been measured using the pulse-radiolysis technique. The temperature dependence of the reaction at low ionic strength yields an activation energy of 31 +/- 5 kJ/mol as compared to 14 +/- 3 kJ/mol for the reaction of CO2.(-) under the same conditions. The pH dependence fits the single pK'a of ferricytochrome c of 9.1. The bimolecular rate constant for the reaction of the superoxide anion with ferricytochrome c at pH 7.8, 21 +/- 2 degrees C, in the presence of 50 mM phosphate and 0.1 mM EDTA is (2.6 +/- 0.1) X 10(5) M-1 s-1. Using this value, 1 unit of superoxide dismutase activity (McCord, J. M., and Fridovich, I. (1969) J. Biol. Chem. 244, 6049-6055) is calculated to be 3.6 +/- 0.3 pmol of enzyme if the assay is performed in a total volume of 3.0 ml. Copper ions reduce the yield of the reaction of ferricytochrome c with CO2.(-). The reactivities of native and singly modified 4-carboxy-2,4-dinitrophenyllysine cytochromes c towards the superoxide anion radical are in the order native greater than 4-carboxy-2,4-dinitrophenyllysine 60 greater than lysine 13 greater than lysine 87 greater than lysine 27 greater than lysine 86 greater than lysine 72, indicating that electron transfer takes place at or close to the solvent accessible heme edge. The mechanism of the reaction is discussed in terms of the approach of superoxide anion radicals to the heme edge and the available molecular orbitals of both heme and free radicals.
    • Kinetics and possible regulation of crypt cell populations under normal and stress conditions.

      Potten, Christopher S; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester (1975)
      The proliferative organisation of the crypts of the small intestine is considered with special reference to the existence, location and numbers of stem cells. It is concluded that the crypt contains a minority population of cells at its base that are the true stem cells. These cells provide an input of cells for the larger proliferative compartment higher up the crypt. The presumptive stem cells may be pluripotent and produce Paneth, goblet and columnar cells. They are probably also the cells which are capable of regenerating the crypt after X-ray depopulation. Radiobiological experiments indicate that the number of cryptogenic cells is less than 80, while the results of several experiments on the kinetics of the cell populations indicate that the number of stem cells is about 20. The stem cells are located in the Paneth cell zone of the crypt, and are apparently passing through the cell cycle at about half the speed of the proliferative cells. It is these vital stem cells that will determine the response of the mucosa to therapeutic agents, probably play a role in carcinogenesis and play a dominant role in mechanisms controlling cell proliferation.
    • Kinetics of cell replacement in the stratum granulosum of mouse tongue epithelium.

      Hume, W J; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Withington, Manchester M20 9BX, U.K. (1986-03)
      Sheet preparations of the stratum granulosum from the epithelium of the ventral surface of mouse tongue permit examination of cell replacement of this maturation compartment of the tissue. The cell transit rate/day is related to the cell desquamation rate and the cell production rate. The latter is approximately 6500-8000 cells/mm2/day, suggesting a 4-5-fold greater turnover compared with mouse dorsal skin epithelium. The use of [3H]IUdR and [3H]TdR at different times of day provides evidence for a reutilization of label from [3H]TdR released during nuclear degradation in the stratum granulosum. Flooding with unlabelled thymidine is not effective in suppressing this reutilization.
    • Kinetics of growth of haemopoietic colony cells in agar.

      Testa, Nydia G; Lord, Brian I; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester (1973-07)
    • The kinetics of hematopoietic stem cells during and after hypoxia. A model analysis.

      Loeffler, M; Herkenrath, P; Wichmann, H E; Lord, Brian I; Murphy, M J; Medizinische Universitätsklinik, Joseph-Stelzmann-Stasse 9, D-5000 Köln 41, Federal Republic of Germany. (1984-12)
      A previously described mathematical model of the hematopoietic stem cell system has been extended to permit a detailed understanding of the data during and after hypoxia. The model includes stem cells, erythroid and granuloid progenitors and precursors. Concerning the intramedullary feedback mechanisms two basic assumptions are made: 1) The fraction "a" of CFU-S in active cell cycle is regulated. Reduced cell densities of CFU-S, progenitors or precursors lead to an accelerated stem cell cycling. Enlarged cell densities suppress cycling. 2) The self renewal probability "p" of CFU-S is also regulated. The normal steady state is described by p = 0.5, indicating that on statistical average each dividing mother stem cell is replaced by one daughter stem cell, while the second differentiates. Diminished cell densities of CFU-S or enlarged densities of progenitors and precursors induce a more intensive self renewal (p greater than 0.5), such that the stem cell number increases. The self renewal probability declines (p less than 0.5) if too many CFU-S or too few progenitors and precursors are present. The model reproduces bone marrow data for CFU-S, BFU-E, CFU-C, CFU-E, 59 Fe-uptake and nucleated cells in hypoxia and posthypoxia. Although the ratio of differentiation into the erythroid and granuloid cell lines is kept constant in the model, a changing ratio of CFU-E and CFU-C results. The model suggests that stem cells and progenitor cells are regulated by a regulatory interference of erythropoiesis and granulopoiesis.
    • Kinetics of neutrophil production in normal and neutropenic animals during the response to filgrastim (r-metHu G-CSF) or filgrastim SD/01 (PEG-r-metHu G-CSF).

      Lord, Brian I; Woolford, Lorna B; Molineux, Graham; CRC Experimental Haematology Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester M20 4BX, United Kingdom. blord@picr.man.ac.uk (2001-07)
      Filgrastim G-CSF has a short, biologically active half-life, and its effective use depends on repeated inoculations. A major aim, therefore, has been to develop a once-per-chemotherapy cycle formulation. To this end, a polyethylene glycolylated form of Filgrastim, known as SD/01, has been developed. In this study, we compared the cellular kinetics of granulocyte production in mice stimulated with SD/01 and granulocyte colony-stimulating factor (G-CSF). Mice were injected with a single dose of SD/01 (1 mg/kg) or G-CSF (125 microg/kg) twice per day for 4 days. Mice rendered leukopenic with a single injection of cyclophosphamide (200 mg/kg) and temozolomide (90 mg/kg) were similarly treated at their 3-day neutrophil nadir. Tritiated thymidine was injected for autoradiographic labeling studies. Bone marrow labeling indices and the release of labeled neutrophils and monocytes into the peripheral blood were assessed. Granulocytopoiesis was stimulated similarly by both SD/01 and G-CSF in both normal and neutropenic animals, with counts rising to >20 x 10(9) polymorphonuclear neutrophils/l in both cases. Bone marrow thymidine labeling indices were increased, indicating a greater proportion of cells in DNA synthesis and an elevated proliferative activity. Compared with the normally slow release of neutrophils into the peripheral blood, labeled neutrophils (and monocytes) were rapidly released, increasing to peak levels at approximately 24 h. The peripheral half-life of neutrophils was not significantly different from normal, and the mitotic amplification factors for increase in granulocytopoiesis, accounted for by 3-3.9 extra cell divisions, were comparable for both factors. We conclude that neutrophil kinetics are stimulated in the same way and to the same extent by both SD/01 and G-CSF.
    • Kinetics of the recognizable erythrocyte precursor cells.

      Lord, Brian I; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester (1979-06)
    • KRAS induces lung tumorigenesis through microRNAs modulation.

      Shi, Lei; Middleton, J; Jeon, Y; Magee, Peter; Veneziano, D; Laganà, A; Leong, Hui Sun; Sahoo, Sudhakar; Fassan, M; Booton, R; et al. (2018-02-13)
      Oncogenic KRAS induces tumor onset and development by modulating gene expression via different molecular mechanisms. MicroRNAs (miRNAs) are small non-coding RNAs that have been established as main players in tumorigenesis. By overexpressing wild type or mutant KRAS (KRASG12D) and using inducible human and mouse cell lines, we analyzed KRAS-regulated microRNAs in non-small-cell lung cancer (NSCLC). We show that miR-30c and miR-21 are significantly upregulated by both KRAS isoforms and induce drug resistance and enhance cell migration/invasion via inhibiting crucial tumor suppressor genes, such as NF1, RASA1, BID, and RASSF8. MiR-30c and miR-21 levels were significantly elevated in tumors from patients that underwent surgical resection of early stages NSCLC compared to normal lung and in plasma from the same patients. Systemic delivery of LNA-anti-miR-21 in combination with cisplatin in vivo completely suppressed the development of lung tumors in a mouse model of lung cancer. Mechanistically, we demonstrated that ELK1 is responsible for miR-30c and miR-21 transcriptional activation by direct binding to the miRNA proximal promoter regions. In summary, our study defines that miR-30c and miR-21 may be valid biomarkers for early NSCLC detection and their silencing could be beneficial for therapeutic applications.