The landscape of metabolic pathway dependencies in cancer cell lines

The metabolic reprogramming of cancer cells creates metabolic vulnerabilities that can be therapeutically targeted. However, our understanding of metabolic dependencies and the pathway crosstalk that creates these vulnerabilities in cancer cells remains incomplete. Here, by integrating gene expression data with genetic loss-of-function and pharmacological screening data from hundreds of cancer cell lines, we identified metabolic vulnerabilities at the level of pathways rather than individual genes. This approach revealed that metabolic pathway dependencies are highly context-specific such that cancer cells are vulnerable to inhibition of one metabolic pathway only when activity of another metabolic pathway is altered. Notably, we also found that the no single metabolic pathway was universally essential, suggesting that cancer cells are not invariably dependent on any metabolic pathway. In addition, we confirmed that cell culture medium is a major confounding factor for the analysis of metabolic pathway vulnerabilities. Nevertheless, we found robust associations between metabolic pathway activity and sensitivity to clinically approved drugs that were independent of cell culture medium. Lastly, we used parallel integration of pharmacological and genetic dependency data to confidently identify metabolic pathway vulnerabilities. Taken together, this study serves as a comprehensive characterization of the landscape of metabolic pathway vulnerabilities in cancer cell lines.     

The occurrence of the Entner-Doudoroff pathway in bacteria

The occurrence of the two key enzymes of the Entner-Doudoroff pathway, gluconate-6-phosphate dehydrase and 2-keto-3-deoxygluconate-6-phosphate aldolase, was determined in approximately 150 strains belonging to 37 different bacterial genera. The following results were obtained:

1. 24 out of 37 genera have at least one representative with the Entner-Doudoroff mechanism. It is thus more widespread than previously thought.
2. The Entner-Doudoroff mechanism occurs mainly in gram-negative bacteria with a DNA base composition in the range 52–70% GC. Eighty-five per cent of these organisms contain the system, while only 20% (6 strains) of the gram-negative organisms with less than 52% GC possess both enzymes.
3. This pathway is absent in all gram-positive organisms investigated except in 5 out of 12Nocardia strains.
4. Erwinia and some strains of theAchromobacter-Alcaligenes group are exceptional, since they possess only 2-keto-3-deoxygluconate-6-phosphate aldolase.

     

Recent trends in cell substrate considerations for continuous cell lines

Product development activity in the past five to ten years has reconstituted a version of an old debate on the safety assessment of biological products, namely whether the use of some types of continuous cell lines (CCLs) is appropriate in the preparation of some types of biological products. Since 1987, dozens of purified recombinant DNA products derived from CCLs have been developed and have received regulatory approval. In addition, several live attenuated and inactivated viral vaccines manufactured in CCLs were approved after thorough review of product safety and manufacturing issues. The current discussion revolves around the potential use of CCLs (human or not) to prepare purified protein subunit vaccines, such as for HIV, and the use of human CCLs to prepare purified protein products.     

Complex chromosome aberrations in continuous mammalian cell lines

Complex chromosome aberrations (CCA) are described, occurring spontaneously in low frequency, in numerous mammalian cell lines. These aberrations appear similar to those reported in leukocyte cultures of some Yanamama Indians. In some cell lines the frequency of CCA is increased by the administration of cytochalasin B (CB) a drug which prevents cytoplasmic division. The frequency of CCA may also be increased by the protease inhibitor tosyl lysyl chloro methyl ketone (TLCK). TLCK may also produce binucleate cells but unlike CB does not result in high degrees of multinucleation. In one cell line, 3T12, the simultaneous administration of CB and TLCK resulted in high frequencies of CCA. Thus the induction of CCA in cell culture is reproducible. However the etiology of CCA remains unknown.     

Genetic analysis of metabolic cooperation in mammalian cell lines

Metabolites can be exchanged between cells in culture by direct transfer from the cytoplasm of one cell to that of another in a process known as metabolic cooperation. Most mammalian cell lines are able to transfer small molecules directly between adjacent cells in this way and are consequently mec+; however, a small number are defective in this ability (mec-). Results obtained from somatic cell hybrids formed between combinations of these cells have shown that the four different cell lines examined in this study can be divided into at least two different complementation groups on the basis of their ability to transfer 3H-labeled nucleotides to adjacent cells. Two of the cell lines clearly fall into a single complementation group.     

The reduction of starch accumulation in transgenic sugarcane cell suspension culture lines

Starch only occurs in small amounts in sugarcane, but is, nevertheless an unwanted product because it reduces the amount of sucrose that can be crystallized from molasses. In an attempt to reduce the starch content of sugarcane, the activities of ADP-glucose pyrophosphorylase (AGPase) and beta-amylase were manipulated using transgenic approaches. Transformation vectors to reduce AGPase activity and to increase plastidial beta-amylase activity were constructed and used for the transformation of sugarcane calli. The results of the manipulations were analyzed in suspension cultures. AGPase activity was reduced down to between 14 and 54% of the wild-type control. This led to a reduction in starch concentration down to 38% of the levels of the wild-type control. beta-Amylase activity was increased in the transgenic lines by 1.5-2 times that of the wild-type control. This increase in activity led to a reduction in starch amounts by 90% compared to wild-type control cells. In both experiments, the changes in starch concentrations could be correlated with the change in enzyme activity. There were no significant effects on sucrose concentrations in either experiment, indicating that these approaches might be useful to engineer regenerated sugarcane for optimized sucrose production.     

The occurrence of the Leloir pathway in non-pathogenic mycobacteria

It has been found that saprophytic strains of mycobacteria can utilize D-galactose via the Leloir pathway which involves galactokinase, galactose-1-phosphate uridyl transferase and UDP-galactose-4-epimerase. The resulted glucose-1-phosphate is further converted by phosphoglucomutase to glucose-6-phosphate and the latter catabolized in glycolitic cycle to pyruvate. The particular enzymes of the galactose pathway have been fully separated by chromatography on a DEAE-cellulose column and some of them partially characterized.     

The metabolic defect of methionine dependence occurs frequently in human tumor cell lines

Methionine dependence is the inability of cells to grow when methionine (Met) is replaced by its immediate precursor homocysteine (Hcy) in the culture medium (Met^?Hcy⁺ medium). All normal unestablished cell strains tested to date have been shown to be methionine-independent and thus grow almost as well in Met^?Hcy⁺ medium as they do in Met⁺Hcy^? medium. Results presented here indicate that out of 23 cell lines derived from diverse types of human tumors, 11 do not grow at all in Met^?Hcy⁺ medium and are absolutely methionine-dependent and 3 grow only slightly in this medium. Many of the tumor cell lines tested have little else in common other than the fact that they are methionine-dependent. The high frequency of occurrence of methionine dependence in diverse types of human tumor cells indicates that methionine dependence may be an important aspect of oncogenic transformation and therapeutically exploitable.     

The acceptability of continuous cell lines: A personal & historical perspective

In the 1950s, only primary cell cultures were acceptable for the production of human biological products. This position was challenged in the late 1960s by human diploid cells (HDCs), and again in the 1980s by continuous cell lines (CCLs). The history of the HDC controversy is reviewed and lessons from that era that are relevant to the use of CCLs are pointed out. It became apparent in the early days of recombinant DNA technology in the 1980s that CCLs were needed for the development of some products. CCL acceptability therefore became more urgent, and several attempts were made to reach a consensus on regulatory issues. In 1986, the World Health Organization convened a Study Group to review the safety issues related to products derived from CCLs. The Study Group made a clear recommendation to pursue CCLs in product development because of the demonstrated capability of modern manufacturing processes to cope with contaminants. Issues such as acceptable levels of cellular DNA in products and the relationship of purity to safety are discussed in the context of the need for regulatory authorities, industry, and the general biomedical community to cooperate in addressing problems in a rational scientific manner.     

Ecdysteroids induce morphological changes in continuous cell lines of Lepidoptera

Summary In the continuous cell lines of the LepidopteraTrichoplusia ni (TN-368) andSpodoptera littoralis (HPB-SL-26), ecdysone and 20-hydroxyecdysone cause a series of morphological changes: some cells aggregate, elongate and extend long thin processes. The extent and rapidity of changes are dose dependent, 20-hydroxy-ecdysone being much more active then ecdysone. The continuous cell lines ofS. littoralis (UIV-SL-573) andS. frugiperda (IPLB-SF-21) were much less responsive to the presence of the ecdysteroids.     

Karyological study of the continuous cell lines. Comparative analysis of the Hela and Detroit-6 cell lines]

Comparison of the results of the karyologic analysis of two Hela cell sublines (HeLa1 and HeLa2), obtained from different sources, and of Detroit-6 cell line has shown that all the lines contain marker chromosomes characteristic of the HeLa cell line. Detroit-6 cell line marker chromosomes are similar to markers of the HeLa subline (HeLa1). At the same time, part of marker chromosomes in the two sublines of HeLa cell line (HeLa1 and HeLa2) are different. These data show that HeLa1 and Detroit-6 cell lines are more similar than two sublines of the same HeLa cell line.     

The ultrastructure of imaginal disc cells in primary cultures and during cell aggregation in continuous cell lines

We have examined the ultrastructure of cellular vesicles in primary cultures of wing imaginal disc cells of Drosophila melanogaster. These cells maintain the apico-basal polarity characteristic of epithelial cells. The apical surfaces secrete extracellular material into the lumen of the vesicle from plasma membrane plaques at the tip of microvilli. During the course of one passage, cells from the established cell lines grow to confluence and then aggregate into discrete condensations joined by aligned bridges of cells. Cells in these aggregates are tightly packed, and there appears to be a loss of the epithelial polarity characteristic of the vesicle cells. Elongated cell extensions containing numerous microtubules are found in aggregates, and we suggest that these may be epithelial feet involved in the aggregation process. Virus particles are commonly found both within the nucleus and the cytoplasm of cells in the aggregates.     

A novel metabolic pathway for leukotriene B4 in different cell types: primary reduction of a double bond

Addition of leukotriene B4 together with trace amounts of tritiated leukotriene B4 to different cell types, such as bone marrow-derived macrophages, T-lymphocytes, mesangial cells or fibroblast tumor cells, led to the formation of several hitherto unknown degradation products within hours. None of them could be identified as 20-hydroxy- or 20-carboxyleukotriene B4, known to be produced by polymorphonuclear leukocytes. The primarily formed transient leukotriene B4 metabolite was less polar than leukotriene B4 and was detectable by measuring its ultraviolet absorbance at 232 nm or its radioactivity. Mass spectral analysis showed very similar fragmentation spectra of leukotriene B4 and its primary metabolite. The most abundant ion and the main fragments of the new metabolite were increased by two mass units compared to leukotriene B4. These observations suggest that, in a variety of cells, leukotriene B4 is first reduced to a 5,12-dihydroxyeicosatrienoic acid, which is further converted to secondary hydrophilic degradation products. This raises the question of the major route of leukotriene B4 metabolism in vivo.     

Variability of continuous insect cell lines and their identification

Continuous insect cell lines make a special object of research in biology. Insect cells in the established lines differ in the number of attributes from both normal differentiated, and embryonic cells. The period of genome destabilization necessarily precedes cell line immortalization. Genome destabilization is manifested by changes in genome size, cell karyotype, amplification of some retrotransposone families, and induction of their expression. The existence of significant genetic variability in one line puts a problem of searching for invariant attributes providing culture identification and defining the limits of normal polymorphism of cells in the culture. Using the vast collection of insect continuous cell lines stored at the N. I. Vavilov Institute of General Genetics RAS, nine lines were identified by RFLP method of mitochondrial DNA. Variability of DNA-polymorphisms, cellular karyology, morphology, immunological and biochemical attribute in the culture is discussed.