Richard P. Hill

Hypoxia and metabolism: Hypoxia, DNA repair and genetic instability

Areas of hypoxic tumour tissue are known to be resistant to treatment and are associated with a poor clinical prognosis. There are several reasons why this might be, including the capacity of hypoxia to drive genomic instability and alter DNA damage repair pathways. Significantly, current models fail to distinguish between the complexities of the hypoxic microenvironment and the biological effects of acute hypoxia exposures versus longer-term, chronic hypoxia exposures on the transcription and translation of proteins involved in genetic stability and cell survival. Acute and chronic hypoxia might lead to different biology within the tumour and this might have a direct effect on the design of new therapies for the treatment of hypoxic tumours.

Exploring the role of cancer stem cells in radioresistance

Radiobiological research over the past decades has provided evidence that cancer stem cell content and the intrinsic radiosensitivity of cancer stem cells varies between tumours, thereby affecting their radiocurability. Translation of this knowledge into predictive tests for the clinic has so far been hampered by the lack of methods to discriminate between stem cells and non-stem cells. New technologies allow isolation of cells expressing specific surface markers that are differentially expressed in tumour cell subpopulations that are enriched for cancer stem cells. Combining these techniques with functional radiobiological assays holds the potential to elucidate the role of cancer stem cells in radioresistance in individual tumours, and to use this knowledge for the development of predictive markers for optimization of radiotherapy.

Carnitine palmitoyltransferase 1C promotes cell survival and tumor growth under conditions of metabolic stress

Tumor cells gain a survival/growth advantage by adapting their metabolism to respond to environmental stress, a process known as metabolic transformation. The best-known aspect of metabolic transformation is the Warburg effect, whereby cancer cells up-regulate glycolysis under aerobic conditions. However, other mechanisms mediating metabolic transformation remain undefined. Here we report that carnitine palmitoyltransferase 1C (CPT1C), a brain-specific metabolic enzyme, may participate in metabolic transformation. CPT1C expression correlates inversely with mammalian target of rapamycin (mTOR) pathway activation, contributes to rapamycin resistance in murine primary tumors, and is frequently up-regulated in human lung tumors. Tumor cells constitutively expressing CPT1C show increased fatty acid (FA) oxidation, ATP production, and resistance to glucose deprivation or hypoxia. Conversely, cancer cells lacking CPT1C produce less ATP and are more sensitive to metabolic stress. CPT1C depletion via siRNA suppresses xenograft tumor growth and metformin responsiveness in vivo. CPT1C can be induced by hypoxia or glucose deprivation and is regulated by AMPKα. Cpt1c-deficient murine embryonic stem (ES) cells show sensitivity to hypoxia and glucose deprivation and altered FA homeostasis. Our results indicate that cells can use a novel mechanism involving CPT1C and FA metabolism to protect against metabolic stress. CPT1C may thus be a new therapeutic target for the treatment of hypoxic tumors.

A Lung-colony Assay to Determine the Radiosensitivity of the Cells of a Solid Tumour

SummaryAn assay system is described in which a number of single tumour cells are injected intravenously into recipient mice and grow in the lungs of these mice to give macroscopic tumour nodules in 16 to 20 days. The relationship between the number of tumour nodules observed and the number of cells injected has been found to be linear up to a mean of 50 colonies/lung for both non-irradiated and irradiated cells, and the inter-experiment consistency has been shown to be good. The number of tumour nodules observed has been found to be affected by the simultaneous injection of a large number of heavily-irradiated cells, provided they are injected intravenously. An aerobic radiation survival-curve has been established and agrees very well with one obtained for the same tumour using the end-point dilution assay method. The assay has the advantage that the time required for completion is short, compared with that required for end-point dilution assays.

pVHL modification by NEDD8 is required for fibronectin matrix assembly and suppression of tumor development

ABSTRACT Functional inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is the cause of the familial VHL disease and most sporadic renal clear-cell carcinomas (RCC). pVHL has been shown to play a role in the destruction of hypoxia-inducible factor α (HIF-α) subunits via ubiquitin-mediated proteolysis and in the regulation of fibronectin matrix assembly. Although most disease-causing pVHL mutations hinder the regulation of the HIF pathway, every disease-causing pVHL mutant tested to date has failed to promote the assembly of the fibronectin matrix, underscoring its potential importance in VHL disease. Here, we report that a ubiquitin-like molecule called NEDD8 covalently modifies pVHL. A nonneddylateable pVHL mutant, while retaining its ability to ubiquitylate HIF, failed to bind to and promote the assembly of the fibronectin matrix. Expression of the neddylation-defective pVHL in RCC cells, while restoring the regulation of HIF, failed to promote the differentiated morphology in a three-dimensional growth assay and was insufficient to suppress the formation of tumors in SCID mice. These results suggest that NEDD8 modification of pVHL plays an important role in fibronectin matrix assembly and that in the absence of such regulation, an intact HIF pathway is insufficient to prevent VHL-associated tumorigenesis.

The proportion of stem cells in murine tumors

Considerable evidence suggests that tumors contain only a minority of cells which are capable of regrowing the tumor (ie. tumor stem cells). Since all tumor stem cells must be killed if treatment is to be successful, the number of stem cells in a tumor can be expected to be an important determinant of curability. We have attempted to examine the proportion of stem cells in a variety of murine tumors by making measurements of three different parameters which might be expected to be related to stem cell content: (a) the radiation dose required to control the tumor (TCD50); (b) the number of cells required to transplant the tumor (TD50) and (c) the in vitro plating efficiency. An inverse correlation has been demonstrated between measured TCD50 and TD50 values for two independent groups of murine tumors of varying histopathological type. An inverse correlation was also obtained between the TD50 value and in vitro plating efficiency for a group of spontaneous murine mammary tumors. These correlations most likely reflect underlying differences in the stem cell content of the tumors, and indicate that there is a wide range (2-3 orders of magnitude) of stem cell proportions in different murine tumors, even those which have been transplanted a number of times.

An examination of the effects of hypoxia, acidosis, and glucose starvation on the expression of metastasis-associated genes in murine tumor cells

Tumor cells exposed to a growth stress such as low pH, glucose starvation and hypoxia have been shown to exhibit a transient increase in experimental metastatic potential, particularly when allowed to recover under normal growth conditions for a period of 24-48 h. In this study we examined whether this increase in metastatic ability could be explained by changes in the expression of a number of different metastasis-associated genes, when the cells were exposed to similar conditions (24-48h exposure to the stress condition followed by 0-48h recovery under normal growth conditions). Although the cell lines used (KHT fibrosarcoma, SCC VII squamous cell carcinoma, and B16F1 melanoma) demonstrated altered metastatic ability after the treatment, no overall temporal correlation between changes in the mRNA levels for cathepsin B, cathepsin L, nm23, TIMP-1, osteopontin, or VEGF and metastatic ability in the three cell lines was observed. The production of gelatinase A (72 kDa collagenase) and gelatinase B (92 kDa collagenase) was also measured by gelatin zymography. There was an increase in production of these enzymes with increasing recovery time, but it did not parallel changes in metastatic potential. Although these results suggest that the products of most of the genes studied may not be involved in the transient metastatic changes, further studies are required to establish whether changes in protein levels track with changes in mRNA levels for these genes.

Analysis of Genomic Integrity and p53-Dependent G1 Checkpoint in Telomerase-Induced Extended-Life-Span Human Fibroblasts

ABSTRACT Life span determination in normal human cells may be regulated by nucleoprotein structures called telomeres, the physical ends of eukaryotic chromosomes. Telomeres have been shown to be essential for chromosome stability and function and to shorten with each cell division in normal human cells in culture and with age in vivo. Reversal of telomere shortening by the forced expression of telomerase in normal cells has been shown to elongate telomeres and extend the replicative life span (H. Vaziri and S. Benchimol, Curr. Biol. 8:279–282, 1998; A. G. Bodnar et al., Science 279:349–352, 1998). Extension of the life span as a consequence of the functional inactivation of p53 is frequently associated with loss of genomic stability. Analysis of telomerase-induced extended-life-span fibroblast (TIELF) cells by G banding and spectral karyotyping indicated that forced extension of the life span by telomerase led to the transient formation of aberrant structures, which were subsequently resolved in higher passages. However, the p53-dependent G1 checkpoint was intact as assessed by functional activation of p53 protein in response to ionizing radiation and subsequent p53-mediated induction of p21Waf1/Cip1/Sdi1. TIELF cells were not tumorigenic and had a normal DNA strand break rejoining activity and normal radiosensitivity in response to ionizing radiation.

The importance of the pre-irradiation breathing times of oxygen and carbogen (5% CO2: 95% O2) on the in vivo radiation response of a murine sarcoma.

Abstract The effect of localized radiation on the transplantable KHT sarcoma in unanesthetized C3H mice was investigated while the mice breathed either oxygen (O 2 ) or 5% CO 2 : 95% O 2 (carbogen) for times ranging from 0.5 to 120 min prior to irradiation. Tumors were irradiated with a single dose of 2000 rad at a dose rate of 1140 rad/min and tumor cell survival was determined using a lung colony assay. The results for O 2 breathing indicate that survival varies as a function of pre-irradiation breathing time (PIBT). Whereas 10 and 30 min give survival values three times lower than air survival, 15–20 min and very long times (1–2 hr) yield results which are similar to those obtained under air breathing conditions. In animals breathing carbogen there is a minimum in the survival level (factor of three lower than air survival) after 10 min of gas exposure followed by a slow rise in survival over the next 90 min at which time there is no sensitization by this gas mixture relative to air. During a course of seven 500 rad fractions given daily with carbogen, a 5 min PIBT was found to give significantly greater sensitization than a 0.5 min PIBT. These findings give a clear indication that the PIBT of O 2 and carbogen has a strong influence on the effectiveness of the radiation treatment and suggest the need to control this factor carefully in the clinic.

Expression of hypoxia-inducible factor-1α in cervical carcinomas: correlation with tumor oxygenation ☆

PURPOSE To investigate the relations between hypoxia-inducible factor-1 (HIF-1), tumor oxygenation, and clinical correlates in patients with locally advanced carcinoma of the uterine cervix. METHODS AND MATERIALS Biopsies from 42 patients with invasive cervical carcinoma and previous polarographic O2 measurements were assessed for the expression of HIF-1alpha using digitized microscopic imaging and analysis. RESULTS The HIF-1alpha expression levels ranged from <0.1% to 10.7% of the total tumor area; the positive staining was localized exclusively to the nuclei. Three distinct arrangement patterns of HIF-1alpha-positive cells in relation to blood vessels were identified using spatial image mapping: (1) most HIF-1alpha-positive cells were located within the typical oxygen diffusion distance in tissue (< or =150 microm to the nearest blood vessel); (2) most HIF-1alpha-positive cells were located in the vicinity (< or =60 microm) of the blood vessels; and (3) no apparent spatial relationship was found between HIF-1alpha-positive cells and blood vessels. A statistically significant association was found between HIF-1alpha expression and tumor oxygenation (Spearman correlation coefficient = 0.4, p <0.01), as determined with the Eppendorf pO2 histograph. No correlation was found between the level of HIF-1alpha expression and patient outcome, using disease-free survival as the end point. CONCLUSION Our results suggest that HIF-1alpha expression may represent a useful biologic marker for hypoxia in uterine cervical cancer.