Loss of the tumor suppressor Pten promotes proliferation of Drosophila melanogaster cells in vitro and gives rise to continuous cell lines

In vivo analysis of Drosophila melanogaster has enhanced our understanding of many biological processes, notably the mechanisms of heredity and development. While in vivo analysis of mutants has been a strength of the field, analyzing fly cells in culture is valuable for cell biological, biochemical and whole genome approaches in which large numbers of homogeneous cells are required. An efficient genetic method to derive Drosophila cell lines using expression of an oncogenic form of Ras (Ras(V12)) has been developed. Mutations in tumor suppressors, which are known to cause cell hyperproliferation in vivo, could provide another method for generating Drosophila cell lines. Here we screened Drosophila tumor suppressor mutations to test if they promoted cell proliferation in vitro. We generated primary cultures and determined when patches of proliferating cells first emerged. These cells emerged on average at 37 days in wild-type cultures. Using this assay we found that a Pten mutation had a strong effect. Patches of proliferating cells appeared on average at 11 days and the cultures became confluent in about 3 weeks, which is similar to the timeframe for cultures expressing Ras(V12). Three Pten mutant cell lines were generated and these have now been cultured for between 250 and 630 cell doublings suggesting the life of the mutant cells is likely to be indefinite. We conclude that the use of Pten mutants is a powerful means to derive new Drosophila cell lines.     

Action of genotypical surrounding of host Drosophila melanogaster on biological effects of endosymbiont Wolbachia (strain wMelPop)

In this work, a comparative study of the structure of symbiotic bacteria Wolbachia (strain wMelPop decreasing the fly lifespan) in genotypically different Drosophila melanogaster, as well as the effect of the bacteria on the host cell ultrastructure was investigated out. As a result of special crossings, the Drosophila melanogaster [w]Trl ³⁶² and [w]Trl ^en82 lines, which are carried of mutations for the gene Trithorax-like, are synthesized (lines infected with Wolbachia are designated as [w]). The Drosophila melanogaster line free of Wolbachia was obtained by treatment with antibiotics of the initially infected [w]w ¹¹¹⁸ line. The complex of the used methods and approaches has allowed us to perform a comparative study of the morphology of cell structures for the first time before and after the infestation of insects with bacteria and to evaluate effect of the bacteria on viability and fertility of flies of these lines. Electron microscopy analysis has shown that the embryos of the analyzed lines contain typical Wolbachia in contact with various host cell compartments; the ultrastructural organization of the bacteria indicates the preservation of their functional activity. In the cytoplasm of embryos that are mutant for the gene Trithorax-like, morphologically atypical mitochondria were revealed, as well as Wolbachia (wMelPop) of unusual morphology with a modified form of membtane envelopes. The presence of Wolbachia in ovarian cells of the female mutant fly lines has been found to produce no effect on the amount of the female-ovipositioned eggs. It has been established for the first time that lifespans of the infected and Wolbachia-free Drosophila melanogaster mutant lines TM3 containing chromosome 3 as a balancer are equal. However, it is significantly shorter in the imago of the [w]w ¹¹¹⁸ line than in flies of the mutant lines. This has allowed us to suggest that either the chromosome-balancer TM3 or mutation of the gene Trl play an important role in the host-symbiont interactions. On checking this suggestion, it was found that the lifespan of homozygotes [w]Trl ³⁶² and [w]Trl ^en82 after the infection of flies with bacteria decreased markedly and was close to the lifespan of [w]w ¹¹¹⁸ line. The obtained data indicate that the chromosome-balancer TM3 can have a significant effect on the symbiont-host interaction.     

The molecular basis of emetine resistance in chinese hamster ovary cells: Alteration in the 40S ribosomal subunit

The molecular basis of resistance to the protein synthesis inhibitor emetine has been examined in cell-free, protein-synthesizing extracts derived from normal and emetine-resistant (Emt^R) mutants. We had earlier shown that protein synthesis in extracts of the mutant cells was resistant to the inhibitory action of the emetin. When extracts from a wild-type and mutant cell line were fractionated into supernatant (S-100) and polyribosome fractions and mixed in different combinations, resistance to emetine was found to be associated with the mutant polyribosome fraction. Further fractionation of wild-type and mutant polyribosomes into 40S and 60S ribosomal subunits and mixing them in various combinations with an S-100 fraction from the wild-type cell indicates that resistance of mutant cells to emetine involves an alteration in the 40S ribosomal subunit.The behavior of Emt^R has also been examined in somatic cell hybrids. Studies of Emt^R × Emt^S hybrid cell lines in vivo and in vitro show that Emt^R is phenotypically recessive to Emt^S, which is consistent with the ribosomal location of the genetic change.     

Rudimentary mutants ofDrosophila melanogaster

Summary The X-linkedrudimentary (r) mutants ofDrosophila melanogaster are pyrimidine auxotrophs and require exogenous pyrimidines (Nørby, 1970; Falk, 1976). We have established a set ofrudimentary cell lines that are derived from embryos, homozygous for eitherr ¹ orr ³⁶. The enzymatic activities of the pyrimidine synthesizing enzymes were measured in the mutant lines. We have further investigated the nutritional requirements of the mutant cells in vitro by using a pyrimidine free culture medium.Ther ¹ cell lines were found to express 3–7%dihydroorotase (DHOase) activity as compared to a wildtype cell line. Reducedaspartate transcarbamylase (ATCase) activity was measured in somer ¹ cell lines whereas wildtypecarbamylphosphate synthetase (CPSase) activity is expressed in allr ¹ cell lines. Ther ³⁶ cell line expresses wildtype activity ofDHOase andCPSase. ATCase activity was found to be reduced to 10% of the wildtype activity.The mutant cell lines do not proliferate in pyrimidine free minimal medium and cell proliferation is obtained by the addition of crude RNA. Proliferation of ther ¹ cells is restored by the supplementation of the minimal medium withdihydroorotate whereas proliferation of ther ³⁶ cells is restored by supplementation with eitherdihydroorotate orcarbamylaspartate.The results demonstrate that therudimentary phenotypesr ¹ andr ³⁶ are expressed at the cellular level and that the two mutant cell types behave as cellular pyrimidine auxotrophs in vitro.     

Mutagen sensitivity of Drosophila melanogaster. I. Isolation and preliminary characterization of a methyl methanesulphonate-sensitive strain

Sensitivity to the monofunctional alkylating agent methyl methanesulfonate (MMS) has been tested as a selection technique to isolate mutant strains which can provide insights into the genetic control of DNA replication, DNA repair and recombination in the complex eucaryote, Drosophila melanogaster. The successful isolation of an X-linked MMS-sensitive strain, mut^s, has suggested that mutagen sensitivity is a feasible methodology for the selection of mutant strains of Drosophila which will be useful in the genetic and biochemical analysis of these cellular functions. Preliminary characterization of this mutant strain indicates that: (A) it is extremely sensitive to killing by MMS; (B) it is more mutable by MMS than the parent wildtype strain; and (C) it appears to possess mutator gene activity.     

2D gene expression parameters of wing imaginal disc of Drosophila for developmental analysis

 By using high resolution two-dimensional (2D) gel electrophoresis coupled with computer-analysis we have established a quantitative Drosophila wing imaginal disc protein database of third instar larvae as a reference to be used for comparative purposes in genetic studies. A general catalogue integrated by 1,184 ³⁵S-methionine-labelled polypeptides from wing imaginal disc has been obtained. The level of expression for all the proteins has been quantitatively determined. The quantitative reproducibility of the analysis system has been estimated and all the controls studied as database reference to interpret the results of experiments with mutant discs. One example, corresponding to iro ¹ mutation, has been used to show how some of the changes observed with mutant discs clearly extend out of the limits defined by the controls. This enables us to generate comparative parameters for the study of proliferation, morphogenesis and differentiation of Drosophila and opens the possibility of rapidly defining the nature and quantity of changes in patterns of gene expression in developmental genetic studies. Received: 21 June 1996 / Accepted: 27 September 1996     

Evolution of karyotype in haploid cell lines of Drosophila melanogaster

Seven continuous cell lines have been established in vitro from lethal embryos produced by the female sterile mutant mh 1182 of Drosophila melanogaster. Six lines show haploid metaphases. Karyotype analysis revealed a high level of aneuploid cells with frequent chromosome fragments. In three lines, haploid cells were quickly overgrown by diploid cells. Two lines were more stable but the proportion of haploid cells decreased with time. One line was stable, showing 80-90% of haploid cells for over 1 000 cell generations. Stable haploid clones have been isolated from two lines. Crossing of mh 1182/mh 1182 females with males bearing a ring X chromosome shows that the haploid genome retained in the cells is of maternal origin and that the diploid cells derive from pre-existing haploid cells. The appearance of the diploid cells and the conditions of karyotypic stability are analysed.     

Genetic evidence that aphidicolin inhibits in vivo DNA synthesis in Chinese hamster ovary cells

Using a genetic approach, Chinese hamster ovary (CHO) cells sensitive (aph^S) and resistant (aph^R) to aphidicolin were grown in the presence or absence of various DNA polymerase inhibitors, and the newly synthesized DNA isolated from [³²P]dNMP-labelled, detergent-permeabilized cells, was characterized after fractionation by gel electrophoresis. The particular aph ^Rmutant CHO cell line used was one selected for resistance to aphidicolin and found to possess an altered DNA polymerase of the a-family. The synthesis of a 24 kb replication intermediate was inhibited in wild-type CHO cells grown in the presence of aphidicolin, whereas the synthesis of this replication intermediate was not inhibited by this drug in the mutant CHO cells or in the aphidicolin-resistant somatic cell hybrid progeny constructed by fusion of wild-type and mutant cell lines. Arabinofuranosylcytosine (ara-C), like aphidicolin, inhibited the synthesis of this 24 kb DNA replication intermediate in the wild-type CHO cells but not in the aph^R mutant cells. However, carbonyldiphosphonate (COMDP) inhibited the synthesis of the 24 kb replication intermediate in both wild-type and mutant cells. N²-(p-n-Butylphenyl)-2 deoxyguanisine-5-triphosphate (BuPdGTP) was found to inhibit the formation of Okazaki fragments equally well in the wild-type and mutant cell lines and thus led to inhibition of synthesis of DNA intermediates in both cases. It appears that aphidicolin and ara-C both affect a common target on the DNA polymerase, which is different from that affected by COMDP in vivo. These data also show that aphidicolin, ara-C and COMDP affect the elongation activity of DNA polymerase but not the initiation activity of the enzyme during DNA replication. This is the first report of such differentiation of the DNA polymerase activities during nuclear DNA replication in mammalian cells. The method of analysis described here for replication intermediates can be used to examine the inhibitory activities of other chemicals on DNA synthesis.     

High efficiency of a double-screening method on single P-element insertion lines to identify quantitative trait mutants in Drosophila melanogaster

Enhancer trap P-element insertion has become a common method for generating new mutations in Drosophila melanogaster. When this method is used to isolate mutants for quantitative traits, an appropriate control must be established to define normal and mutant phenotypes. Considering that enhancer-trap lines are generated by crossing several strains, usually with no homogeneous genetic background, no clear control strain can be selected. Previous reports tried to overcome this problem by homogenizing the genetic background of the original lines. However, this is not the most common scenario, especially when functional phenotypes are studied in previously generated lines. Without such caution, is it possible to identify functional mutants among P-element insertion lines? We tested this for olfactory preference, a quantitative trait. Using as control measurement the average phenotype of 30 simultaneously generated P-element insertion lines with preferential reporter-gene expression in olfactory reception organs, we found that 25 of the lines exhibited mutant phenotypes in response to one or several of 5 tested odorants. Additional tests showed that the efficiency of the method for detecting olfactory mutations exceeded 60% even for such a small number of tested odorants. According to these results this approach greatly facilitates the identification of putative abnormal phenotypes, which must be extensively confirmed afterwards.     

CD4: p56lck Association Studied in vivo Using Antibody-Induced Capping and Double Indirect Immunofluorescence Microscopy

Abstract

Accumulating data suggest that the T-cell surface antigen CD4 transduces an independent signal during antigen-mediated T-cell activation. In vitro studies which showed that the cytoplasmic protein tyrosine kinase p56^lck is present in anti-CD4 immunoprecipitates led to the model that p56^lck is associated with the cytoplasmic domain of CD4. In this report we have extended these studies and examined potential CD4:p56^lck associations in vivo. We show here by double immuno-fluorescence microscopy a specific co-distribution of p56^lck with antibody-induced CD4 caps in intact cells. Murine T-cell hybridoma lines expressing mutant forms of CD4 were used to demonstrate that the 31 carboxyterminal aminoacids of its cytoplasmic domain, in particular cysteine-420 and cysteine-422, are crucial for the formation of CD4:p56^lck complexes in vivo. The potential of the method applied is discussed with regard to studies of other transmembrane signalling systems involving src-like kinases.     

Efficient genetic method for establishing Drosophila cell lines unlocks the potential to create lines of specific genotypes

Analysis of cells in culture has made substantial contributions to biological research. The versatility and scale of in vitro manipulation and new applications such as high-throughput gene silencing screens ensure the continued importance of cell-culture studies. In comparison to mammalian systems, Drosophila cell culture is underdeveloped, primarily because there is no general genetic method for deriving new cell lines. Here we found expression of the conserved oncogene Ras(V12) (a constitutively activated form of Ras) profoundly influences the development of primary cultures derived from embryos. The cultures become confluent in about three weeks and can be passaged with great success. The lines have undergone more than 90 population doublings and therefore constitute continuous cell lines. Most lines are composed of spindle-shaped cells of mesodermal type. We tested the use of the method for deriving Drosophila cell lines of a specific genotype by establishing cultures from embryos in which the warts (wts) tumor suppressor gene was targeted. We successfully created several cell lines and found that these differ from controls because they are primarily polyploid. This phenotype likely reflects the known role for the mammalian wts counterparts in the tetraploidy checkpoint. We conclude that expression of Ras(V12) is a powerful genetic mechanism to promote proliferation in Drosophila primary culture cells and serves as an efficient means to generate continuous cell lines of a given genotype.     

The thymidine-kinase locus of friend erythroleukaemic cells: 1. Mutation rates and properties of mutants

Spontaneous mutation at the thymidine-kinase locus in clone 707 of the Friend cell lines has been examined. The rate of mutation in BrdU resistance was found to be 2.6 × 10^?6 cell^?1 generation^?1. The rate of reversion to HAT resistance was found to vary from 1.1 × 10^?7 to 2.85 × 10^?6 cell^?1 generation^?1 in 4 BrdU-resistant clones. Of 14 mutant clones assayed for thymidine-kinase activity only one had greater than 13% the activity of wild-type cells. Fluctuation analysis showed that mutations occurred spontaneously and were not induced by the selective agent.The mutation rate at the thymidine-kinase locus is several orders of magnitude greater than those reported for several other cell lines. There does not appear to be a general genetic instability in this cell line as the mutation rate at the HGPRT locus is similar to those found in other established cell lines.It is suggested that the high forward mutation rate at the thymidine-kinase locus may be due to only one functional thymidine-kinase allele being present in cells of this alone.     

Alterations in both acetylcholinesterase activity and synaptic scaffolding protein localization in the nervous system of Drosophila presenilin mutants

Presenilins are one of two types of critical genetic factors in familial Alzheimer's disease, and they regulate various cellular functions such as intracellular Ca²⁺ homeostasis, the endoplasmic reticulum (ER) stress response, apoptosis, and synaptic transmission. We utilized Drosophila presenilin (psn) mutants as a model for studying the role of this gene in regulating acetylcholinesterase activity (AChE) and synaptic plasticity. Several lines of biochemical evidence indicated that AChE activity in a functionally null psn mutant (psn^B3) was significantly reduced. In addition, we also found that psn^B3 mutant neuromuscular junctions (NMJs) had smaller synaptic boutons and altered localization of Discs large, a synaptic scaffolding protein at the synaptic terminals compared to wild-type controls. These phenotypic defects were completely rescued in transgenic lines expressing the long form of wild-type Psn under an endogenous psn promoter cassette (PEPC-Psn^WT;psn^B3 lines). Taken together, these results indicate that Psn is important for regulating AChE activity, the size of synaptic boutons, and the localization of DLG at synaptic terminals.     

A complementation method for functional analysis of mammalian genes

Our progress in understanding mammalian gene function has lagged behind that of gene identification. New methods for mammalian gene functional analysis are needed to accelerate the process. In yeast, the powerful genetic shuffle system allows deletion of any chromosomal gene by homologous recombination and episomal expression of a mutant allele in the same cell. Here, we report a method for mammalian cells, which employs a helper-dependent adenoviral (HD-Ad) vector to synthesize small hairpin (sh) RNAs to knock-down the expression of an endogenous gene by targeting untranslated regions (UTRs). The vector simultaneously expresses an exogenous version of the same gene (wild-type or mutant allele) lacking the UTRs for functional analysis. We demonstrated the utility of the method by using PRPF3, which encodes the human RNA splicing factor Hprp3p. Recently, missense mutations in PRPF3 were found to cause autosomal-dominant Retinitis Pigmentosa, a form of genetic eye diseases affecting the retina. We knocked-down endogenous PRPF3 in multiple cell lines and rescued the phenotype (cell death) with exogenous PRPF3 cDNA, thereby creating a genetic complementation method. Because Ad vectors can efficiently transduce a wide variety of cell types, and many tissues in vivo, this method could have a wide application for gene function studies.     

A point mutation (G574A) in the chemokine receptor CXCR4 detected in human cancer cells enhances migration

The chemokine receptor CXCR4 is widely expressed in human cancers and regulates cell invasion, proliferation and survival. Because mutations in the CXCR4 gene could regulate its function we sequenced the coding region of the CXCR4 gene in 18 human melanoma and 3 human colon carcinoma cell lines. The same somatic point mutation (G574A; V160I) in the fourth trans-membrane region of CXCR4 was detected in one colon cancer cell line (PD) and one melanoma cell line (LB). CXCR4 was expressed and functional in both PD and LB cells, PD and LB cells migrated specifically toward the receptor ligand, CXCL12 and P-Erk was specifically induced by CXCL12. To give insight into the function of the mutant CXCR4 receptor, human A431, epidermoid carcinoma cells, were stably transfected with both mutant and wild type CXCR4. In vitro, A431 cells harboring CXCR4^G574A migrated specifically toward CXCL12 and CXCL12 induced ERK phosphorylation. Interestingly, in vivo studies showed that the growth of A431 tumors harboring CXCR4G574A was delayed compared to those harboring WT CXCR4. As expected, treatment with AMD3100, a specific CXCR4 inhibitor, reduced the in vivo growth of CXCR4_^G574A tumor b^G574A rprisingly, increased the growth of CXCR4^G574A A431 cells. This is the first report of a spontaneously occurring, functionally active CXCR4 mutation in human cancer cells. While the mutation impairs cell growth in vivo, the CXCR4 inhibitor, AMD3100, stimulated the growth of cells harboring CXCR4^G574A.     

Inherited deficiencies of human methylmalonyl CaA mutase activity: reduced affinity of mutant apoenzyme for adenosylcobalamin.

We have studied the affinity of methylmalonyl CoA mutase for its required cofactor, adenosylcobalamin, in extracts of control and mutant human cultured fibroblasts. Control enzyme has an apparent K_m for adenosylcobalamin of 6–7 × 10^?8 M. Five mutant cell lines from patients with methylmalonicacidemia due to a mutase apoenzyme defect were studied. Three have undetectable mutase activity (<0.15% of control) at all cofactor concentrations. Two others, however, have markedly altered K_m's for adenosylcobalamin of 2.8 × 10^?4 M and 1.7 × 10^?5 M. These mutant lines synthesize adenosylcobalamin normally and, by complementation analysis, are genetically identical to all other mutase apoenzyme mutants tested. We conclude that the mutase deficiency in these two cell lines results from structurally altered mutase apoenzymes with markedly reduced affinities for adenosylcobalamin.     

Evidence for intrachromosomal gene conversion in cultured mouse cells

In this report we present an experimental scheme that facilitates the study of homologous recombination between closely linked genes in cultured mammalian cells. Two different Xho I linker insertion mutants of the herpes simplex virus type 1 thymidine kinase (HTK) gene were introduced into mouse LTK^? cells as direct repeats on a plasmid carrying a dominant selectable marker. Following stabilization of these sequences in the recipient cell, selection for TK⁺ was applied to detect recombinational events between different TK^? genes. TK⁺ segregants were observed at a frequency of 10^?4–10^?5 in lines harboring both mutant genes. Control lines carrying only one type of mutant HTK gene yielded TK⁺ cells at frequencies of 10^?7 or less. Physical analysis of the TK⁺ segregants has revealed the presence of an apparently normal HTK gene that is resistant to Xho I endonuclease digestion in each TK⁺ line examined. Analyses of the TK gene pairs before and after recombination suggest that at least 50% of the recombinants are the result of nonreciprocal exchanges of genetic information, or gene conversion events.     

To Cease or To Proliferate: New Insights into TCTP Function from a Drosophila Study

Tor (target of rapamycin) pathway underlies a major signaling mechanism for controlling cell growth and proliferation.¹ Rheb (Ras homolog enriched in brain) is a small GTPase in the Tor pathway.^2–4 Similar to other small GTPases, Rheb cycles between a GTP-bound active state and a GDP-bound inactive state. TSC2 (tuberous sclerosis complex 2), a gene mutated in an autosomal dominant disease tuberous sclerosis, was shown to be the Rheb-GAP (GTPase activating protein).^5,6 However, a guanine nucleotide exchange factor (GEF) for Rheb had been missing. Human TCTP (translationally controlled tumor protein) has been implicated in cancer, but its function in vivo has not been clearly elucidated. Recently we reported a molecular genetic characterization of TCTP function in Drosophila.⁷ Drosophila TCTP (dTCTP) displays GEF activity to Rheb and is essential for Rheb activation in organ growth. Thus, our study provides a tight linkage of dTCTP to the Rheb-TOR pathway. In this addendum, we will briefly overview our findings and discuss our perspectives for future research on TCTP.Key Words: TCTP, Tor pathway, Rheb, TSC, GEFTCTP is a highly conserved protein identified about 20 years ago as a translationally controlled protein P21 (or P23) enriched in tumor cell lines.⁸ Recently, this protein has drawn special interests because of its potential roles in tumorigenesis. TCTP is not only upregulated in a number of tumor cell lines but also downregulated during tumor reversion.^9,10 TCTP has also been implicated in a variety of intracellular and extracellular functions, including microtubule stabilization, cell cycle, apoptosis, and cytokine release.^11–16 However, these functions of TCTP have been inferred mainly from biochemical interactions and cell culture studies.To address the function of TCTP in vivo, we took a loss-of-function approach using Drosophila as a genetic model. Reduction of dTCTP by tissue-specific RNA interference (RNAi) or loss-of-function mutations resulted in smaller organs with reduction in both cell size and cell number, a phenotype often seen in mutations in Tor or insulin pathway. Our epistatic analysis suggested that dTCTP acts either downstream or in parallel to insulin receptor, TSC1, and dRheb, but upstream of dS6K. Despite the conserved sequence of TCTP proteins in a wide-range of species, TCTP has little similarity to the sequences of other protein families. However, the three-dimensional structure of fission yeast TCTP ortholog reveals similarities with a family of proteins that bind to the nucleotide-free form of Rab GTPases,¹⁷ providing a clue for its potential biochemical function. Consistent with our genetic evidence, our biochemical assays showed that dTCTP could facilitate the GDP/GTP exchange on dRheb both in vitro and in vivo. Our data led us to propose a model (Fig. 1) in which dTCTP regulates the Tor signaling pathway by directly interacting with dRheb GTPase as a GEF.Open in a separate windowFigure 1A model for dTCTP function in growth control. dRheb GTPase stimulates Tor signaling, which in turn activates dS6K and CycE to regulate cell growth and proliferation, respectively. dRheb GTPase is inactivated by the GAP function of TSC1/2 complex. In contrast, dTCTP activates dRheb GTPases by promoting GDP-GTP exchange. dTCTP might have additional functions independent of the Tor pathway, such as inhibiting cell death. It remains to be determined if dTCTP regulates cell proliferation and cell survival in part through the Hippo signaling.This study not only provides new insights into the mechanism of dTCTP function in regulation of dRheb activity but also reveals a complexity of dTCTP function in growth regulation. Firstly, dTCTP null mutant displays more severe phenotypes than loss of function mutations in the insulin or Tor pathways. For example, while dTCTP null mutant clones are eliminated during development, significant portions of dRheb and Tor null mutant clones can survive to form adult tissues. This argues against the model that dTCTP functions only in regulating the Rheb-Tor pathways. Although different maternal contribution of each gene and variations in genetic background might partially account for these differences, it is equally possible that dTCTP functions as a GEF for more than one GTPase targets. Conversely, other GEFs might exist for Rheb, as one small GTPase can be regulated by more than one GEFs.^18,19 Different GEFs might be required for dRheb-TOR function in other developmental events or growth-independent processes like axon guidance during neural development.²⁰Secondly, in contrast to the implicated role of TCTP in cancer,⁹ ubiquitous or tissue-specific overexpression of dTCTP was insufficient to cause overgrowth phenotypes in Drosophila. A plausible explanation is that the amount of dTCTP is in excess for dRheb activation in contrast to limited concentrations of TSC2 GAP in normal cells.⁵ Since overexpression of dTCTP alone does not result in tumorous overgrowth, it is unlikely that TCTP functions as an oncogene directly under normal condition. However, increased amounts of TCTP in tumor tissues may provide better potency for cells to undergo uncontrolled proliferation and massive overgrowth. In this regard, it will be intriguing to learn if dTCTP can act corporately with other oncogenes. Since the amino acid sequence identity of dTCTP and human TCTP is only 48%, it is also possible that a non-conserved region(s) of human TCTP may be required to induce tumors in human cells. Soon after the publication of our work, knockout of the mouse TCTP was reported. Remarkably, loss of TCTP in mice results in early lethality with smaller sizes of embryos.²¹ Developmental defects seen in mutant mice may be analogous to the phenotypes of dTCTP mutants in Drosophila, supporting the conserved function of TCTP in growth regulation. However, it is yet to be determined whether there is a common molecular basis for the developmental defects in both systems and whether overexpression of mouse TCTP in transgenic mice can induce tumors.Lastly, in addition to Tor and insulin signaling pathways, a third pathway consisting of Hippo-Warts protein kinase cascade controls organ size by affecting mainly the cell number.^22,23 The Hippo pathway regulates both cell proliferation and cell death by promoting cyclin E expression and downregulating Drosophila Inhibitor of Apoptosis 1 (DIAP1). It is worthy of note that dTCTP also regulates the cell number by affecting cell proliferation and apoptosis. It remains to be determined whether dTCTP and Hippo signaling pathways crosstalk or are independent of each other.Studies on TCTP suggest that its function is much more complex than what is known and its interactions with a multitude of proteins might underlie this complexity. Biochemical studies have identified several proteins interacting with TCTP. Given the feasibility of Drosophila genetics, the physiological relevance of these protein interactions can now be addressed in the context of normal development. It would also be powerful to take the advantage of Drosophila genetic screens for the identification of novel genes interacting with dTCTP. A more comprehensive understanding of TCTP functions and mechanistic explanations of its intriguing expression profiles in cancers can be expected in years to come.