Microtubule Acetylation Controls MDA-MB-231 Breast Cancer Cell Invasion through the Modulation of Endoplasmic Reticulum Stress.

During aggressive cancer progression, cancer cells adapt to unique microenvironments by withstanding various cellular stresses, including endoplasmic reticulum (ER) stress. However, the mechanism whereby cancer cells overcome the ER stress to survive remains to be elucidated. Herein, we demonstrated that microtubule acetylation in cancer cells grown on a stiff matrix promotes cancer progression by preventing excessive ER stress. Downregulation of microtubule acetylation using shRNA or CRSIPR/Cas9 techniques targeting ATAT1, which encodes α-tubulin N-acetyltransferase (αTAT1), resulted in the upregulation of ER stress markers, changes in ER morphology, and enhanced tunicamycin-induced UPR signaling in cancer cells. A set of genes involved in cancer progression, especially focal adhesion genes, were downregulated in both ATAT1-knockout and tunicamycin-treated cells, whereas ATAT1 overexpression restored the gene expression inhibited by tunicamycin. Finally, the expression of ATAT1 and ER stress marker genes were negatively correlated in various breast cancer types. Taken together, our results suggest that disruption of microtubule acetylation is a potent therapeutic tool for preventing breast cancer progression through the upregulation of ER stress. Moreover, ATAT1 and ER stress marker genes may be useful diagnostic markers in various breast cancer types.

Pharmacophore anchor models of ATAT1 to discover potential inhibitors and lead optimization.

Post-translation modification of microtubules is associated with many diseases like cancer. Alpha Tubulin Acetyltransferase 1 (ATAT1) is a major enzyme that acetylates 'Lys-40' in alpha-tubulin on the luminal side of microtubules and is a drug target that lacks inhibitors. Here, we developed pharmacophore anchor models of ATAT1 which were constructed statistically using thousands of docked compounds, for drug design and investigating binding mechanisms. Our models infer the compound moiety preferences with the physico-chemical properties for the ATAT1 binding site. The results from the pharmacophore anchor models show the three main sub-pockets, including S1 acetyl site, S2 adenine site, and S3 diphosphate site with anchors, where conserved moieties interact with respective sub-pocket residues in each site and help in guiding inhibitor discovery. We validated these key anchors by analyzing 162 homologous protein sequences (>99 species) and over 10 structures with various bound ligands and mutations. Our results were consistent with previous works also providing new interesting insights. Our models applied in virtual screening predicted several ATAT1 potential inhibitors. We believe that our model is useful for future inhibitor discovery and for guiding lead optimization.

Enhanced anticancer effects of a methylation inhibitor by inhibiting a novel DNMT1 target, CEP 131, in cervical cancer.

Methylation is a primary epigenetic mechanism regulating gene expression. 5-aza-2'-deoxycytidine is an FDA-approved drug prescribed for treatment of cancer by inhibiting DNA-Methyl-Transferase 1 (DNMT1). Results of this study suggest that prolonged treatment with 5-aza-2'-deoxycytidine could induce centrosome abnormalities in cancer cells and that CEP131, a centrosome protein, is regulated by DNMT1. Interestingly, cancer cell growth was attenuated in vitro and in vivo by inhibiting the expression of Cep131. Finally, Cep131-deficient cells were more sensitive to treatment with DNMT1 inhibitors. These findings suggest that Cep131 is a potential novel anti-cancer target. Agents that can inhibit this protein may be useful alone or in combination with DNMT1 inhibitors to treat cancer. [BMB Reports 2019; 52(5): 342-347].

Upregulation of SPAG6 in Myelodysplastic Syndrome: Knockdown Inhibits Cell Proliferation via AKT/FOXO Signaling Pathway.

Recently, sperm-associated antigen 6 (SPAG6), a member of the cancer-testis antigen family, has been shown to be involved in tumorigenesis. An increasing number of studies have shown that SPAG6 expression is associated with the pathogenesis of myelodysplastic syndrome (MDS). However, the mechanism has not been clearly elucidated. Our previous results indicated that SPAG6 affected cell apoptosis in MDS. In this study, we used reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to demonstrate that the mRNA expression of SPAG6 in bone marrow cells of patients with MDS or MDS-derived acute myeloid leukemia (MDS-AML) was higher than that of cancer-free patients. Kaplan-Meier survival curve analysis of published AML found that patients with high expression of SPAG6 had poor survival. The results of the cell counting kit-8, FACS, RT-qPCR, and Western blotting assays indicated that SPAG6 knockdown in the SKM-1 cell line inhibited cell proliferation, and affected cell cycle and differentiation. Furthermore, we found that SPAG6 knockdown affected the proliferation of SKM-1 cells by mediating the G1-to-S transition of the cell cycle. Moreover, we demonstrated that the antiproliferative effect of SPAG6 knockdown was associated with the upregulation of the cyclin-dependent kinase inhibitor p27Kip1, and regulation of the AKT/FOXO pathway. These findings indicated that SPAG6 might be a potential therapeutic target.

SPAG6 silencing induces apoptosis in the myelodysplastic syndrome cell line SKM­1 via the PTEN/PI3K/AKT signaling pathway in vitro and in vivo.

Apoptosis is a multi-step mechanism of cell self­destruction for maintaining cellular homeostatic balance. Accumulating evidence indicates that abnormal apoptosis promotes the evolution and progression of myelodysplastic syndromes (MDS). As a novel cancer-testis antigen, sperm­associated antigen 6 (SPAG6) has been reported to regulate apoptosis through the tumor necrosis factor-related apoptosis-inducing ligand signaling pathway in the MDS cell line SKM­1. However, the mechanism of the intrinsic cell death pathway for apoptosis induction by SPAG6 silencing is unclear. In the present study, the in vitro effects of SPAG6 silencing were investigated in SKM­1 cells through extensive biochemical and molecular approaches. Western blotting and reverse transcription-quantitative polymerase chain reaction were used to detect the expression of SPAG6 and activation of PTEN/PI3K/AKT signal pathway. Additionally, SKM­1 cells transduced with SPAG6 short hairpin RNA (shRNA) lentivirus were treated with the phosphatidylionositol 3-kinase (PI3K) inhibitor LY294002 or pan caspase inhibitor z­VAD­fmk and the apoptosis rates were measured by flow cytometry, and the expressions of associated proteins were examined by western blot analysis. A mouse xenograft model was also used to further evaluate the effects of SPAG6 knockdown on inducing tumor apoptosis in vivo. Lentivirus-mediated knockdown of SPAG6 in SKM­1 cells increased phosphatase and tensin homolog (PTEN) expression and reduced protein kinase B (AKT) phosphorylation, which in turn resulted in cell apoptosis as evidenced by induced myeloid leukaemia cell differentiation protein Mcl­1 downregulation, cytochrome c release and increased caspase­9 expression. Consistently, the PI3K inhibitor LY294002 synergistically enhanced apoptosis of SKM­1 cells when co-administered with SPAG6 shRNA lentivirus. Furthermore, treatment with the pan caspase inhibitor z­VAD­fmk failed to prevent PTEN activation upon SPAG6 knockdown, suggesting that SPAG6-regulated PTEN expression was caspase activation-independent. In addition, SPAG6 knockdown was associated with DNMT1 downregulation, implying that SPAG6 may indirectly control PTEN expression via DNA methylation. Furthermore, tumor tissues from nonobese diabetic/severe combined immunodeficient mice inoculated with SPAG6-shRNA lentivirus pre-infected SKM­1 cells exhibited significantly elevated apoptosis in the extrinsic and intrinsic pathways. These results demonstrate that SPAG6 silencing induces PTEN expression to regulate apoptosis though the PI3K/AKT pathway, indicating that SPAG6 may be a potential therapeutic target for MDS.

Synergistic Antiproliferative Activity of the RAD51 Inhibitor IBR2 with Inhibitors of Receptor Tyrosine Kinases and Microtubule Protein.

Although cancer cell genetic instability contributes to characteristics that mediate tumorigenicity, it also contributes to the tumor-selective toxicity of some chemotherapy drugs. This synthetic lethality can be enhanced by inhibitors of DNA repair. To exploit this potential Achilles heel, we tested the ability of a RAD51 inhibitor to potentiate the cytotoxicity of chemotherapy drugs. 2-(Benzylsulfonyl)-1-(1H-indol-3-yl)-1,2-dihydroisoquinoline (IBR2) inhibits RAD51-mediated DNA double-strand break repair but also enhances cytotoxicity of the Bcr-Abl inhibitor imatinib. The potential for synergy between IBR2 and more drugs was examined in vitro across a spectrum of cancer cell lines from various tissues. Cells were exposed to IBR2 simultaneously with inhibitors of receptor tyrosine kinases, DNA-damaging agents, or microtubule disruptors. IBR2, at concentrations that inhibited proliferation between 0% and 75%, enhanced toxicity by up to 80% of imatinib and regorafenib (targets RAF and kit); epidermal growth factor receptor inhibitors erlotinib, gefitinib, afatinib, and osimertinib; and vincristine, an inhibitor of microtubule function. However, IBR2 antagonized the action of olaparib, cisplatin, melphalan, and irinotecan. A vincristine-resistant squamous cell line was not cross resistant to imatinib, but IBR2 and another RAD51 inhibitor (B02) enhanced imatinib toxicity in this cell line, its HN-5a parent, and the colon cancer line HT-29 by up to 60% and much better than verapamil, a P-glycoprotein inhibitor (P < 0.05). Given the disparate agents the functions of which are enhanced by IBR2, the mechanisms of enhancement may be multimodal. Whether RAD51 is common to these mechanisms remains to be elucidated, but it provides the potential for selectivity to tumor cells.

Resveratrol induces dephosphorylation of Tau by interfering with the MID1-PP2A complex.

The formation of paired helical filaments (PHF), which are composed of hyperphosphorylated Tau protein dissociating from microtubules, is one of the pathological hallmarks of Alzheimer's disease (AD) and other tauopathies. The most important phosphatase that is capable of dephosphorylating Tau at AD specific phospho-sites is protein phosphatase 2 A (PP2A). Here we show that resveratrol, a polyphenol, significantly induces PP2A activity and reduces Tau phosphorylation at PP2A-dependent epitopes. The increase in PP2A activity is caused by decreased expression of the MID1 ubiquitin ligase that mediates ubiquitin-specific modification and degradation of the catalytic subunit of PP2A when bound to microtubules. Interestingly, we further show that MID1 expression is elevated in AD tissue. Our data suggest a key role of MID1 in the pathology of AD and related tauopathies. Together with previous studies showing that resveratrol reduces ß-amyloid toxicity they also give evidence of a promising role for resveratrol in the prophylaxis and therapy of AD.

Rapid quantification of a cleavable antibody-conjugated drug by liquid chromatography/tandem mass spectrometry with microwave-assisted enzymatic cleavage.

Antibody-drug conjugates (ADCs) play an increasingly important role for targeted cancer treatment. One class of ADCs has attracted particular interest in drug development. These ADCs employ a cleavable chemistry linkage for drugs and utilize the reduced interchain disulfide cysteine residues for conjugation. In this work, a novel bioanalytical method for the quantification of a cleavable antibody-conjugated drug in plasma was developed, qualified, and implemented. This novel method significantly improves throughput by combining a microwave-assisted, enzymatic cleavage of conjugated drugs from ADCs with a 96-well based sample preparation procedure to immunocapture ADCs in plasma. The released drug is subsequently quantified using a LC/MS/MS method. Our results represent a high-throughput, generic, and sensitive quantification method for antibody-conjugated microtubule inhibitors (such as MMAE) for preclinical PK/PD studies. The linear range of the standard curve for antibody conjugated drug (MMAE) was from 2.01 to 2010ng/mL with an excellent linearity (r(2)>0.997). The intra-run precision was below 8.14% and accuracy was from -7.71% to -1.08%. No matrix effect or carryover was observed for this method. This method was successfully used to measure the level of conjugated drug in a preclinical PK/PD study in mice.

Alpha-catenin-dependent recruitment of the centrosomal protein CAP350 to adherens junctions allows epithelial cells to acquire a columnar shape.

Epithelial morphogenesis involves a dramatic reorganisation of the microtubule cytoskeleton. How this complex process is controlled at the molecular level is still largely unknown. Here, we report that the centrosomal microtubule (MT)-binding protein CAP350 localises at adherens junctions in epithelial cells. By two-hybrid screening, we identified a direct interaction of CAP350 with the adhesion protein α-catenin that was further confirmed by co-immunoprecipitation experiments. Block of epithelial cadherin (E-cadherin)-mediated cell-cell adhesion or α-catenin depletion prevented CAP350 localisation at cell-cell junctions. Knocking down junction-located CAP350 inhibited the establishment of an apico-basal array of microtubules and impaired the acquisition of columnar shape in Madin-Darby canine kidney II (MDCKII) cells grown as polarised epithelia. Furthermore, MDCKII cystogenesis was also defective in junctional CAP350-depleted cells. CAP350-depleted MDCKII cysts were smaller and contained either multiple lumens or no lumen. Membrane polarity was not affected, but cortical microtubule bundles did not properly form. Our results indicate that CAP350 may act as an adaptor between adherens junctions and microtubules, thus regulating epithelial differentiation and contributing to the definition of cell architecture. We also uncover a central role of α-catenin in global cytoskeleton remodelling, in which it acts not only on actin but also on MT reorganisation during epithelial morphogenesis.

SPAG6 silencing inhibits the growth of the malignant myeloid cell lines SKM-1 and K562 via activating p53 and caspase activation-dependent apoptosis.

SPAG6, which is a novel cancer-testis antigen, is overexpressed in myeloid malignancies. Previously, SPAG6 was found in UPD (uniparental disomy) region of myeloid cell DNA from MDS patients and reported that SPAG6 may be a predictive marker of minimal residual disease in pediatric acute myeloid, but the biological role of SPAG6 in myeloid malignancies remains unclear. The present study was undertaken to determine the expression and functional significance of SPAG6 in malignant myeloid hematologic cell lines. A short hairpin RNA (shRNA) targeting SPAG6 was designed that could specifically inhibit SPAG6 expression at the mRNA and protein levels when introduced into the malignant myeloid hematologic cell lines SKM-1 and K562. The results from flow cytometry and CCK-8 assays showed that SPAG6 silencing inhibited the proliferation of SKM-1/K562 by inducing apoptosis. Furthermore, SPAG6 silencing resulted in activation of caspase-3, -9 and -8 and upregulated the mRNA and protein expression of p53 and PTEN. Then, we subcutaneously inoculated the monoclonal cells into NOD/SCID mice to establish xenograft models, and we found that the SPAG6-shRNA lentivirus dramatically inhibited tumor growth and increased apoptosis in vivo. These findings demonstrate that SPAG6 might have a role in malignant myeloid hematologic cell proliferation and apoptosis by regulating caspase proteins and p53, suggesting that SPAG6 may be a potential therapeutic target.

Metformin anti-tumor effect via disruption of the MID1 translational regulator complex and AR downregulation in prostate cancer cells.

BACKGROUND: Metformin is an approved drug prescribed for diabetes. Its role as an anti-cancer agent has drawn significant attention because of its minimal side effects and low cost. However, its mechanism of anti-tumour action has not yet been fully clarified. METHODS: The effect on cell growth was assessed by cell counting. Western blot was used for analysis of protein levels, Boyden chamber assays for analyses of cell migration and co-immunoprecipitation (CoIP) followed by western blot, PCR or qPCR for analysis of protein-protein and protein-mRNA interactions. RESULTS: Metformin showed an anti-proliferative effect on a wide range of prostate cancer cells. It disrupted the AR translational MID1 regulator complex leading to release of the associated AR mRNA and subsequently to downregulation of AR protein in AR positive cell lines. Inhibition of AR positive and negative prostate cancer cells by metformin suggests involvement of additional targets. The inhibitory effect of metformin was mimicked by disruption of the MID1-α4/PP2A protein complex by siRNA knockdown of MID1 or α4 whereas AMPK activation was not required. CONCLUSIONS: Findings reported herein uncover a mechanism for the anti-tumor activity of metformin in prostate cancer, which is independent of its anti-diabetic effects. These data provide a rationale for the use of metformin in the treatment of hormone naïve and castration-resistant prostate cancer and suggest AR is an important indirect target of metformin.

Antimitotic agents of natural origin.

Antimitotic agents have been the most successful pharmacological agents for the treatment of cancer. The term "antimitotic agent" has traditionally been synonymous with tubulin-targeting compounds, but as a consequence of the large number of new compounds and mechanisms that have been identified recently, a much broader definition is currently needed. This review attempts to provide a broad overview of compounds and their cognate protein targets which result in a block in mitosis. Focus has been placed on agents that act directly on the mitotic machinery rather than on targets further upstream such as growth factor receptors.

Microtubule inhibitory effects of various SJ compounds on tissue culture cells.

SJ compounds (SJ8002 and related compounds) are a group of novel anticancer agents (Cho, Chung, Lee, Kwon, Kang, Joo, and Oh. PCT/KR02/00392). To explore the anticancer mechanism of these compounds, we examined the effect of SJ8002 on microtubules of six human cell lines. At a high concentration (2 microg/mL), SJ8002 effectively disrupted microtubules of the six cell lines within 1 h. At lower concentrations (0.05 to approximately 1.0 microg/mL), the antimicrotubule activity of SJ8002 varied defending on cell lines. The inhibition of in vitro polymerization of pure tubulin by SJ8002 suggested that SJ8002 acts on free tubulin, inhibits the polymerization of tubulin dimer into microtubules, and hence induces the depolymerization of microtubules.

A new screening method for antimitotic substances and isolation of glycolipids as stimulators of tubulin polymerization from Okinawan sponge Pseudoceratina sp.

A new screening method to detect antimitotic substances utilizing purified porcine brain microtubule proteins was developed. This method observes the inhibitory and stimulatory activities on microtubule polymerization and inhibitory activity on depolymerization in sequence. Two glycolipids, 1-O-beta-D-galactopyranosyl-2,3-di-O-acylglycerol and 1-O-tetrahydroxycyclopentyl-2-O-acyl-3-O-alkylglycerol were isolated from Okinawan marine sponge Pseudoceratina sp. by this screening method. These compounds stimulated the microtubule polymerization at 10 degrees C.

Involvement of TLCK-sensitive serine protease in colchicine-induced cell death of sympathetic neurons in culture.

Superior cervical ganglion (SCG) cells from neonatal rats underwent apoptosis upon treatment with colchicine, a microtubule-disrupting agent. Western blotting and activity measurements showed that caspase-3 was indeed activated, but its peptide inhibitor (Ac-DEVD-CHO) neither suppressed nuclear fragmentation nor rescued the neurons from cell death. z-VAD-fmk, the general inhibitor of caspases, prevented nuclear fragmentation and delayed the cell death. Moreover, N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK), but not N-alpha-tosyl-L-phenylalanine chloromethyl ketone (TPCK), prevented nuclear fragmentation and provided neuronal protection as well. The combination of both z-VAD-fmk and TLCK provided a long-term neuronal protection (>4 days), whereas neither one alone could do so, suggesting that there are both caspase-dependent and -independent pathways. TLCK-sensitive serine protease is also likely to act upstream of caspase-3 in a caspase-dependent pathway. Electron microscopic observations demonstrated that z-VAD-fmk suppressed nuclear fragmentation and improved mitochondrial swelling, but failed to prevent vesicular formation, which resulted in a slowly-occurring necrosis. More importantly, TLCK effectively blocked this abundant vesicular formation along with suppressing chromatin condensation. Thus, the combination of both conferred a nearly normal morphology, which is consistent with the results of cell survival experiments. These findings clearly indicate that TLCK-sensitive serine protease plays multiple roles in caspase-dependent and -independent pathways of colchicine-induced cell death, and suggest a novel mechanism underlying a necrotic pathway involving ER swelling and vesicular formation.

AP-1 activation and altered AP-1 composition in association with increased phosphorylation and expression of specific Jun and Fos family proteins induced by vinblastine in KB-3 cells.

Vinblastine and other microtubule inhibitors are important antitumor agents that cause mitotic arrest, and induce apoptosis through poorly understood mechanisms, in a wide variety of cell lines. The activating protein 1 (AP-1) transcription factor is a major target of the c-Jun NH2-terminal kinase (JNK) signaling pathway, which is activated by microtubule inhibitors. Therefore, we examined the effect of vinblastine on AP-1 composition and activity in human KB-3 carcinoma cells. Vinblastine caused highly selective effects on AP-1 proteins in a concentration- and time-dependent manner. Specifically, c-Jun, expressed at a low level in control cells, was greatly increased and phosphorylated, Jun D was phosphorylated, Jun B underwent phosphorylation and subsequently became undetectable, and Fra 1 expression was also greatly increased. In contrast. Fra 2, c-Fos, and Fos B were relatively unchanged by vinblastine. Changes in AP-1 preceded caspase 3 activation and, therefore, occurred prior to the commitment phase of apoptosis. With the exception of c-Jun, which was not affected by paclitaxel, the same alterations in AP-1 proteins occurred after exposure to vincristine, paclitaxel, and colchicine, demonstrating that these are general responses to microtubule inhibition. Supershift assays demonstrated that in control cells, AP-1 binding activity was mediated by Jun D/Fra 2 heterodimers, whereas after vinblastine treatment, AP-1 complexes also containing c-Jun and Fra 1 were present, suggesting that induction of these latter proteins by vinblastine is functionally significant. Consistent with these observations, vinblastine stimulated AP-1-dependent luciferase reporter gene transcription. These findings suggest that alterations in AP-1 composition and activity may be key events in the early response of KB-3 cells to microtubule inhibitors.

Decreasing oncoprotein 18/stathmin levels reduces microtubule catastrophes and increases microtubule polymer in vivo.

Oncoprotein 18/stathmin (Op18) has been identified recently as a protein which destabilizes microtubules. To characterize the function of Op18 in living cells, we used microinjection of anti-Op18 antibodies or antisense oligonucleotides to block either Op18 activity or expression in interphase newt lung cells. Anti-tubulin staining of cells microinjected with anti-Op18 and fixed 1-2 hours after injection showed an increase in total microtubule polymer. In contrast, microinjection of either non-immune IgG or anti-Op18 preincubated with bacterially-expressed Op18 had little effect on microtubule polymer level. Cells treated with Op18 antisense oligonucleotides for 4 days had (greater than or equal to)50% reduced levels of Op18 with no change in the soluble tubulin level. Measurement of MT polymer level in untreated, antisense or nonsense oligonucleotide treated cells demonstrated that reduced Op18 levels resulted in a 2.5-fold increase in microtubule polymer. Next, the assembly dynamics of individual microtubules at the peripheral regions of living cells were examined using video-enhanced contrast DIC microscopy. Microinjection of antibodies against oncoprotein 18 resulted in a 2.2-fold reduction in catastrophe frequency and a slight reduction in plus end elongation velocity compared to uninjected cells or cells microinjected with non-immune IgG. Preincubation of anti-Op18 antibody with recombinant Op18 greatly diminished the effects of the antibody. Similarly, treatment of cells with antisense oligonucleotides reduced catastrophes 2.5- to 3-fold compared to nonsense oligonucleotide treated or untreated cells. The other parameters of dynamic instability were unchanged after reducing Op18 with antisense oligonucleotides. These studies are consistent with Op18 functioning to regulate microtubule catastrophes during interphase in vivo.

Anti-invasive activities of experimental chemotherapeutic agents.

We have discussed a number of agents that affect invasion and we have grouped them according to their most probable targets. This strategy is based on the following hypothesis. Invasion is the result of cellular responses to extracellular signals. Candidate signals are components of the extracellular matrix, which are rendered inactive by the flavonoid (+)-catechin (see Section III). Signals are recognized by receptors on the plasma membrane, possibly glycoproteins, that may lose their recognition function through alteration of the oligosaccharide side chains by inhibitors of protein glycosylation (see Section IV) and possibly also by alkyllysophospholipids (see Section V). Synthetic oligopeptides reflecting sequences from cell-binding domains of extracellular matrix molecules are also effective tools for blocking specific receptors (see Section VI). GTP-binding proteins (G proteins) act as signal transducers and can be inactivated by pertussis toxin (see Section VII). An intriguing aspect of both alkyllysophospholipids and pertussis toxin is that they can either inhibit the invasion of constitutively invasive cells or induce invasion of constitutively noninvasive cells. Without doubt, cellular responses implicated in invasion are many-fold. Discussed here are cell motility and directional migration with inhibition through dipyridamole and its analogs and through microtubule inhibitors, respectively (see Section VIII). Alternative hypotheses and alternative strategies for the dissection of the invasion process do exist, and alternative cellular and molecular mechanisms of action may explain the anti-invasive activity of the agents discussed earlier. The latter are mentioned in each section. It is the authors' opinion that the possibilities for exploiting the battery of anti-invasive agents have by no means been exhausted. Introducing researchers to experiments that may lead to an understanding of the mechanisms of invasion and metastasis and to new rationales for cancer treatment has been the purpose of our review.

Regulation of the microtubule-lysosome interaction: activation by Mg2+ and inhibition by ATP.

We developed a sedimentation assay to characterize and quantify the association of purified lysosomes to reconstituted microtubules (Mithieux, G., Audebet, C. and Rousset. B. (1988) Biochim. Biophys. Acta 969, 121-130). In the present work, we have examined the potential regulatory role of ATP and Mg2+ on the microtubule-lysosome interaction. The formation of microtubule-lysosome complexes takes place in the absence of Mg2+, but is activated by the addition of Mg2+; both the rate of the interaction and the amount of complexes formed are increased. The maximal effect is observed between 1.5 and 3.5 mM free Mg2+. Measured at the plateau of the interaction, the proportion of microtubules bound to lysosomes increases as a function of free Mg2+ concentration; at optimal concentration of free Mg2+, 90% of the microtubules present in the incubation mixture are bound to lysosomes. ATP induces a concentration-dependent inhibition of the formation of microtubule-lysosome complexes. The half-maximal effect is obtained at an ATP concentration of 0.83 +/- 0.11 mM (n = 7). The effect of ATP is not related to ATP hydrolysis, since ATP exerts its inhibitory action in the presence of EDTA. The ATP effect is mimicked by GTP, p[NH]ppA and tripolyphosphate, ADP and pyrophosphate, but not by AMP or phosphate. In the presence of 1 mM ATP, a Mg2+ concentration of 3 mM (corresponding to 2 mM free Mg2+) is required to overcome the inhibition caused by ATP; above 3 mM, Mg2+ exerts its activating effect. Since the modulating effects of ATP and Mg2+ are obtained at concentrations closed to those occurring in intact cells, we conclude that the regulation of the microtubule-lysosome interaction reported in this paper could be of physiological significance.