MicroRNAs: Genomics, Biogenesis, Mechanism, and Function

The hallmarks of cancer.

Recent data have expanded the concept that inflammation is a critical component of tumour progression. Many cancers arise from sites of infection, chronic irritation and inflammation. It is now becoming clear that the tumour microenvironment, which is largely orchestrated by inflammatory cells, is an indispensable participant in the neoplastic process, fostering proliferation, survival and migration. In addition, tumour cells have co-opted some of the signalling molecules of the innate immune system, such as selectins, chemokines and their receptors for invasion, migration and metastasis. These insights are fostering new anti-inflammatory therapeutic approaches to cancer development.

Inflammation and cancer

In contrast to normal differentiated cells, which rely primarily on mitochondrial oxidative phosphorylation to generate the energy needed for cellular processes, most cancer cells instead rely on aerobic glycolysis, a phenomenon termed “the Warburg effect.” Aerobic glycolysis is an inefficient way to generate adenosine 5′-triphosphate (ATP), however, and the advantage it confers to cancer cells has been unclear. Here we propose that the metabolism of cancer cells, and indeed all proliferating cells, is adapted to facilitate the uptake and incorporation of nutrients into the biomass (e.g., nucleotides, amino acids, and lipids) needed to produce a new cell. Supporting this idea are recent studies showing that (i) several signaling pathways implicated in cell proliferation also regulate metabolic pathways that incorporate nutrients into biomass; and that (ii) certain cancer-associated mutations enable cancer cells to acquire and metabolize nutrients in a manner conducive to proliferation rather than efficient ATP production. A better understanding of the mechanistic links between cellular metabolism and growth control may ultimately lead to better treatments for human cancer.

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Understanding the Warburg Effect: The Metabolic Requirements of Cell Proliferation

Microarrays can measure the expression of thousands of genes to identify changes in expression between different biological states. Methods are needed to determine the significance of these changes while accounting for the enormous number of genes. We describe a method, Significance Analysis of Microarrays (SAM), that assigns a score to each gene on the basis of change in gene expression relative to the standard deviation of repeated measurements. For genes with scores greater than an adjustable threshold, SAM uses permutations of the repeated measurements to estimate the percentage of genes identified by chance, the false discovery rate (FDR). When the transcriptional response of human cells to ionizing radiation was measured by microarrays, SAM identified 34 genes that changed at least 1.5-fold with an estimated FDR of 12%, compared with FDRs of 60 and 84% by using conventional methods of analysis. Of the 34 genes, 19 were involved in cell cycle regulation and 3 in apoptosis. Surprisingly, four nucleotide excision repair genes were induced, suggesting that this repair pathway for UV-damaged DNA might play a previously unrecognized role in repairing DNA damaged by ionizing radiation.

/pdf/significance-analysis-of-microarrays-applied-to-the-ionizing-10j4beasgw.pdf

Significance analysis of microarrays applied to the ionizing radiation response

The human cdk2/cyclin A kinase complex is a key regulator of the events of S phase. This complex contains several proteins involved in regulating its catalytic activity, including one or more of the CKS proteins, which have recently been shown to inhibit the activation of the cdk2 kinase. To investigate whether the CKS genes may be altered in human neoplasia, we mapped the chromosome locations of CKS1 and CKS2 by fluorescence in situ hybridization (FISH). CKS1 was localized to 8q21, a locus that is seldom grossly altered in cancer. The localization of CKS2 to 9q22 places it very near to a putative tumour suppressor locus suggested to be responsible for susceptibility to the Basal Cell Nervus Syndrome (BCNS or Gorlin's syndrome) familial cancer disorder. Six fibroblast cell lines isolated from patients with BCNS were demonstrated by FISH to have both copies of CKS2 present. Partial sequencing of a genomic clone of CKS2 revealed that the open reading frame lies over three exons. Examination of the six cell lines by SSCP and PCR-based sequencing of the parts of the three exons coding for the full length protein demonstrated no consistent divergence from the reported cDNA sequence in any exon. It is unlikely that CKS2 is the BCNS tumour suppressor gene.

Chromosomal mapping of the human genes CKS1 to 8q21 and CKS2 to 9q22.

Callibothrium verticillatum, a cestode from the spiral intestine of the dogfish, Mustelus canis, incorporated a characteristic polyunsaturated fatty acid of marine organisms (22:6 03) without conversion or degradation. The tapeworm was incubated in vitro with [14C] 22:6 w3. Its labeled lipids were fractionated and their radioactivities assayed by column and thin-layer radiochromatography nlethods. Triglyceride (50% of dpm collected) and unesterified fatty acids (28%) were the most radioactive fractions. Sterol ester, wax ester, diacylglyceryl ether, diglyceride, monoglyceride, cardiolipin, phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine also were radioactive. 22:6 ca3 and the other fatty acids of the tapeworm were collected by preparative gas-liquid radiochromatography from the total fatty acids, hydrogenated total fatty acids, triglyceride fatty acids, and unesterified fatty acids. Only 22:6 w3 was radioactive. Lipids were 25% of the dry weight of C. verticillatum. The major lipids were triglyceride (54 wt% of total) and phosphatidylcholine (25%). The polyunsaturated fatty acids of the neutral lipids were mostly eicosenoic (20:5 w3 was 25% of total), and of the polar lipids were docosenoic (22:6 w3 was 30%). The fatty acid composition of the tapeworm, the contents of the spiral intestine, and the fluids and tissues of the dogfish were strikingly similar. It is concluded that polyunsaturates may be passed intact from one trophic level to another in marine food chains, and that tapeworms can be used to monitor lipids in the food chains in which the hosts participate. Polyunsaturated C20 and C22 fatty acids are characteristic components of the storage and membrane lipids of most marine organisms; for example, planktonic unicellular algae (except blue-greens and the Chlorophyceae) (Kates and Volcani, 1966; Ackman et al., 1968; Chuecas and Riley, 1969; Holz, 1969; Lee and Loeblich, 1971), benthic multicellular algae (except Chlorophyceae) (Wagner and Pohl, 1965; Chuecas and Riley, 1966; Pohl et al., 1968; Radunz, 1968; Jamieson and Reid, 1972), protozoa (Uronema sp., Labyrinthula sp.) (Harrington, G. W., and Holz, G. G., Jr., unpublished), phycomycete fungi (Ellenbogen et al., 1969), invertebrates (Ackman and Eaton, 1966, 1967; Ackman et al., 1970; Culkin and Morris, 1970a, b; Lee et al., 1971), and vertebrates (Kayama et al., 1963a, b; Ackman, 1967; Ackman et al., 1971; Malins and Wekell, 1970). They are synthesized de novo by the photosynthetic planktonic and benthic algae. Marine animals incorporate them intact from their diet, and also biosynthesize them by elongating and desaturating C18 acids such as 18:2 o6, 18:3 w3, and 18:4 o3. Some invertebrates may obtain C20 and C22 polyunsaturates and/or their precursors from seawater directly Received for publication 20 March 1973. through their body surfaces (Testerman, 1972). Heterotrophic algae, protozoa, and the fungi may supply themselves with the C20 and C22 polyunsaturates by de novo synthesis, by direct incorporation from the environment, and by elongation and desaturation of the Cis fatty acids. The most typical marine polyunsaturates are o)3 eicosapentaenoic and docosahexaenoic acids with methylene-interrupted double bonds of the cis configuration; i.e., all-cis, 5,8,11,14, 17-20:5 and 4,7,10,13,16,19-22:6 (Ackman, 1964; Hinchcliffe and Riley, 1971). The cestodes of sharks contain these fatty acids (Buteau et al., 1969, 1971) and it has been assumed that they acquire them from their immediate environment, the contents of the spiral intestine. We have tested the possibility of direct incorporation, by exposing a tetraphyllidean of sharks to [14C] 22:6 o3 and then performing a radiochemical analysis of the lipids of the tapeworm to determine the distribution of the polyunsaturate. Cestodes were chosen as particularly favorable material for an experiment on direct incorporation of 22:6 oj3 since they neither oxidize nor biosynthesize fatty acids de novo (Meyer et al., 1966).

Incorporation of docosahexaenoic fatty acid into the lipids of a cestode of marine elasmobranchs.

We have identified a novel human gene by virtue of its ability to complement the rad1-1 checkpoint mutant of Schizosaccharomyces pombe. This gene, called RACH2, rescues the temperature-sensitive lethality of a rad1-1 wee1-50 double mutant of S. pombe. Expression of RACH2 in S. pombe rad1-1 strains partially restores UV resistance to the rad1-1 mutant strain. Expression of RACH2 in a rad1-1 cdc25-22 double mutant partially restores the dose-dependent delay in mitotic entry after irradiation that is lost in rad1-1 checkpoint-deficient mutants. Overexpression of RACH2 in human tissue culture cells induces apoptosis.

RACH2, a novel human gene that complements a fission yeast cell cycle checkpoint mutation.

EMX homeobox genes regulate microphthalmia and alter melanocyte biology.

A method of identifying compounds or molecules which alter (enhance or inhibit) stimulation of kinase activity of pre-MPF and, thus, alter (enhance or inhibit) activation of MPF and entry into mitosis. The present method thus makes it possible to identify compounds or molecules which can be administered to regulate the cell cycle; such compounds are also the subject of this invention.

David Beach

Papers

Chromosomal mapping of the human genes CKS1 to 8q21 and CKS2 to 9q22.

Incorporation of docosahexaenoic fatty acid into the lipids of a cestode of marine elasmobranchs.

RACH2, a novel human gene that complements a fission yeast cell cycle checkpoint mutation.

EMX homeobox genes regulate microphthalmia and alter melanocyte biology.

Method of screening for antimitotic compounds using the CDC25 tyrosine phosphatase