scholarly journals Taxol Suppresses Dynamics of Individual Microtubules in Living Human Tumor Cells

1999 ◽  
Vol 10 (4) ◽  
pp. 947-959 ◽  
Author(s):  
Anne-Marie C. Yvon ◽  
Patricia Wadsworth ◽  
Mary Ann Jordan
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
Mitotic Spindle ◽  
Dynamic Instability ◽  
Human Tumor ◽  
Mitotic Checkpoint ◽  
Microtubule Dynamics ◽  
Mitotic Spindles ◽  
Ovarian Adenocarcinoma ◽  
Adenocarcinoma Cells

Microtubules are intrinsically dynamic polymers, and their dynamics play a crucial role in mitotic spindle assembly, the mitotic checkpoint, and chromosome movement. We hypothesized that, inliving cells, suppression of microtubule dynamics is responsible for the ability of taxol to inhibit mitotic progression and cell proliferation. Using quantitative fluorescence video microscopy, we examined the effects of taxol (30–100 nM) on the dynamics of individual microtubules in two living human tumor cell lines: Caov-3 ovarian adenocarcinoma cells and A-498 kidney carcinoma cells. Taxol accumulated more in Caov-3 cells than in A-498 cells. At equivalent intracellular taxol concentrations, dynamic instability was inhibited similarly in the two cell lines. Microtubule shortening rates were inhibited in Caov-3 cells and in A-498 cells by 32 and 26%, growing rates were inhibited by 24 and 18%, and dynamicity was inhibited by 31 and 63%, respectively. All mitotic spindles were abnormal, and many interphase cells became multinucleate (Caov-3, 30%; A-498, 58%). Taxol blocked cell cycle progress at the metaphase/anaphase transition and inhibited cell proliferation. The results indicate that suppression of microtubule dynamics by taxol deleteriously affects the ability of cancer cells to properly assemble a mitotic spindle, pass the metaphase/anaphase checkpoint, and produce progeny.

Mechanically cut mitotic spindles: clean cuts and stable microtubules

1989 ◽  
Vol 94 (3) ◽  
pp. 415-423
Author(s):  
R.B. Nicklas ◽  
G.M. Lee ◽  
C.L. Rieder ◽  
G. Rupp
Keyword(s):  
Electron Microscopy ◽  
Mitotic Spindle ◽  
Dynamic Instability ◽  
Microtubule Dynamics ◽  
Mitotic Spindles ◽  
Mechanical Method ◽  
Spindle Structure ◽  
Main Spindle ◽  
Spindle Body

We have discovered an easy way to cut through the mitotic spindle at any desired place. Spindles of demembranated cricket or grasshopper spermatocytes were severed with a microneedle between the chromosomes and one pole, and the cut-off polar piece was swept away. Spindle structure and microtubule dynamics in cut spindles were studied by anti-tubulin immunostaining and electron microscopy. The cut is clean: all microtubules are severed and only a few extend beyond the others. This provides the basis for a clear test of whether traction fibers pull chromosomes to the pole in anaphase, because the putative traction fiber is cleanly severed. Cutting creates new plus ends on microtubules in the cut-off polar piece and new minus ends on microtubules in the main spindle body. The microtubules with new plus ends are unstable, as expected from the dynamic instability of microtubules. However, the microtubules with new minus ends are as stable as uncut microtubules in the same spindle. Our mechanical method of cutting microtubules very likely creates native, reactive ends, and therefore the surprising stability of new minus ends is genuinely interesting, not an artifact of cutting.

scholarly journals Evidence for a Negative Correlation between Human Reactive Enamine-Imine Intermediate Deaminase A (RIDA) Activity and Cell Proliferation Rate: Role of Lysine Succinylation of RIDA

2021 ◽  
Vol 22 (8) ◽  
pp. 3804
Author(s):  
Luisa Siculella ◽  
Laura Giannotti ◽  
Benedetta Di Chiara Stanca ◽  
Matteo Calcagnile ◽  
Alessio Rochira ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
Negative Correlation ◽  
Cancer Biology ◽  
Human Tumor ◽  
In Silico Analysis ◽  
Proliferation Rate ◽  
Reactive Intermediate ◽  
Cell Proliferation Rate

Reactive intermediate deaminase (Rid) proteins are enzymes conserved in all domains of life. UK114, a mammalian member of RidA subfamily, has been firstly identified as a component of liver perchloric acid-soluble proteins (L-PSP). Although still poorly defined, several functions have been attributed to the mammalian protein UK114/RIDA, including the reactive intermediate deamination activity. The expression of UK114/RIDA has been observed in some tumors, arousing interest in this protein as an evaluable tumor marker. However, other studies reported a negative correlation between UK114/RIDA expression, tumor differentiation degree and cell proliferation. This work addressed the question of UK114/RIDA expression in human non-tumor HEK293 cell lines and in some human tumor cell lines. Here we reported that human RIDA (hRIDA) was expressed in all the analyzed cell line and subjected to lysine (K-)succinylation. In HEK293, hRIDA K-succinylation was negatively correlated to the cell proliferation rate and was under the control of SIRT5. Moreover, K-succinylation clearly altered hRIDA quantification by immunoblotting, explaining, at least in part, some discrepancies about RIDA expression reported in previous studies. We found that hRIDA was able to deaminate reactive enamine-imine intermediates and that K-succinylation drastically reduced deaminase activity. As predicted by in silico analysis, the observed reduction of deaminase activity has been related to the drastic alterations of hRIDA structure inferred by K-succinylation. The role of hRIDA and the importance of its K-succinylation in cell metabolism, especially in cancer biology, have been discussed.

scholarly journals A New Sulfated Triterpene Saponin from Gypsophila trichotoma

2013 ◽  
Vol 8 (6) ◽  
pp. 1934578X1300800 ◽  
Author(s):  
Ilina Krasteva ◽  
Maya Yotova ◽  
Kristina Jenett-Siems ◽  
Petranka Zdraveva ◽  
Stefan Nikolov
Keyword(s):  
Cell Proliferation ◽  
Tumor Cell ◽  
Cell Lines ◽  
Spectral Methods ◽  
Human Tumor ◽  
Malignant Cell ◽  
Tumor Cell Lines ◽  
Human Tumor Cell ◽  
Human Tumor Cell Lines ◽  
Dependent Inhibition

A new sulfated triterpeniod saponin, 3- O-sulfooleanolic acid 28- O-[ β-glucopyranosyl-(1→3)]-[ β-glucopyranosyl-(1→6)]- β-glucopyranosyl ester (1), along with three known Δ7-sterols: stigmast-7-en-3 β-ol (2), stigmast-7-en-3- O-β-D-glucopyranoside (3) and stigmast-7-en-3-on (4) were isolated from the roots of Gypsophila trichotoma Wend. (Caryophyllaceae). Their structures were elucidated by chemical and spectral methods. Compound 1 caused concentration-dependent inhibition of malignant cell proliferation against different human tumor cell lines.

Regulation of Microtubule Assembly: The View From The End

2000 ◽  
Vol 6 (S2) ◽  
pp. 80-81
Author(s):  
L. Cassimeris ◽  
C. Spittle ◽  
M. Kratzer
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Dynamic Instability ◽  
Intrinsic Property ◽  
Microtubule Dynamics ◽  
Microtubule Assembly ◽  
Chromosome Movement ◽  
Spindle Formation ◽  
Associated Proteins

The mitotic spindle is responsible for chromosome movement during mitosis. It is composed of a dynamic array of microtubules and associated proteins whose assembly and constant turnover are required for both spindle formation and chromosome movement. Because microtubule assembly and turnover are necessary for chromosome segregation, we are studying how cells regulate microtubule dynamics. Microtubules are polarized polymers composed of tubulin subunits; they assemble by a process of dynamic instability where individual microtubules exist in persistent phases of elongation or rapid shortening with abrupt transitions between these two states. The switch from elongation to shortening is termed catastrophe, and the switch from shortening to elongation, rescue. Although dynamic instability is an intrinsic property of the tubulin subunits, cells use associated proteins to both speed elongation (∼ 10 fold) and regulate transitions.The only protein isolated to date capable of promoting fast polymerization consistent with rates in vivo is XMAP215, a 215 kD protein from Xenopus eggs.

Spindle Checkpoint Insufficiency in Acute Myeloid Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 873-873
Author(s):  
Dominik Schnerch ◽  
Julia Felthaus ◽  
Monika Engelhardt ◽  
Ralph M. Waesch
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Mitotic Spindle ◽  
Myeloid Leukemia ◽  
Mitotic Checkpoint ◽  
Mitotic Arrest ◽  
Cyclin B ◽  
Resting Cells ◽  
Chromosome Attachment ◽  
Acute Myeloid

Abstract Chromosomal instability and aneuploidy are hallmarks of most human malignancies. Various mechanisms have been shown to give rise to numerical chromosome aberrations. Compromised function of the spindle assembly checkpoint (SAC) is generally regarded as one of the most powerful ways to drive genome instability. The SAC is a mitotic checkpoint mechanism ensuring the equal segregation of the mitotic chromosomes onto the developing daughter cells. Unfaithful mitotic surveillance by the SAC favors chromosomal misdistribution as error-prone chromosome attachment to the mitotic spindle does not induce a strong mitotic arrest by interference with anaphase promoting complex (APC)-dependent proteolysis. The APC is an important ubiquitin ligase that triggers the transition from mitosis into G1-phase by targeted proteolysis of mitotic regulators such as cyclin B and securin. The SAC prevents the proteolysis of those regulator proteins in the presence of mitotic aberrancies by inhibition of the APC. This leads to a delayed progression through mitosis and provides time to recover from defective chromosomal spindle attachment. SAC malfunction weakens the tight control on chromosome attachment and tension across the kinetochore favoring chromosomal misdistribution. We performed expression analyses of key proteins in SAC signaling in acute myeloid leukemia (AML). We found the SAC-components Bub1 and BubR1 to be down-regulated in most of the investigated AML cell lines. Functional assays revealed a defective mitotic arrest mechanism in comparison to SAC-competent cell lines after exposure to the microtubule disrupting agent nocodazole. This finding was accompanied by the observation of a decline in cyclin B and securin levels despite severe damage to the mitotic spindle induced by nocodazole. Expression of cyclin B and securin in the presence of spindle damage could be stabilized by proteasome inhibition. We established a shRNA-based model to evaluate the effects of BubR1- and/or Bub1-repression to levels found among AML cell lines to directly compare the Bub1/BubR1 knockdown phenotype with the investigated AML cell lines. Interestingly, BubR1 knockdown was sufficient to generate a phenotype resembling the behavior of our AML cell lines. Further experiments revealed a strong relation between premature degradation of cyclin B and the degree of BubR1 downregulation. Given the potent role of BubR1 in the generation of a mitotic arrest deficient phenotype, we addressed the BubR1 expression levels in a number of patients exhibiting karyotype abnormalities. Primary myeloid blast cells were stimulated with cytokines to force the largely resting cells into an actively dividing state. The maximum expression level of BubR1 in G2/M was used to define SAC-compentent and SAC-deficient populations. Strikingly, six out of eight (6/8) primary AML samples exhibited BubR1 expression patterns resembling the BubR1-knockdown model suggesting deficient mitotic surveillance in most of the primary AML samples. Since SAC deficiency is an important mechanism in creating numerical chromosomal aberrations and genetic instability, our findings underline a role for impaired SAC function in rise and progression of AML.

The Spindle Checkpoint Protein BubR1 Is Differentially Expressed in the Lymphatic and Myeloid Lineage and Is a Determinator of Response to Spindle Poisons

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1407-1407
Author(s):  
Dominik Schnerch ◽  
Marie Follo ◽  
Andrea Schmidts ◽  
Julika Krohs ◽  
Julia Felthaus ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mitotic Spindle ◽  
Cancer Biology ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Cpg Island ◽  
Mitotic Checkpoint ◽  
Live Cell ◽  
Cyclin B ◽  
Upstream Region

Abstract Abstract 1407 Introduction: Spindle poisons disrupt the mitotic spindle thus leading to activation of the spindle assembly checkpoint (SAC). The SAC is a mitotic surveillance mechanism that can interfere with anaphase-promoting complex/cyclosome- (APC/C-) dependent proteolysis of key cell cycle regulators, such as securin and cyclin B, to delay cells at the metaphase to anaphase transition. The SAC protein BubR1 is a tumor suppressor with critical functions during mitosis and has also been shown to be important for the stablilization of cyclin B during interphase. We found this critical player to be downregulated in AML cell lines, primary AML blast populations and myeloblast-like murine 32D cells when compared to ALL cell lines and lymphoblast-like murine BaF3 cells. While myeloblast-like 32D cells are untransformed cells but also exhibit BubR1 repression we speculate that there might be a physiological role for a reduced BubR1 expression level in healthy myelopoiesis. However, in highly proliferative AML cells BubR1 repression might be a source of genetic instability due to a less efficient SAC-mediated interference with APC/C-dependent proteolysis in the presence of inaccuracies during mitosis. Methods and results: Since repression of BubR1 is known to shorten the metaphase duration, we performed live-cell imaging of leukemia cells and found myeloblastic Kasumi-1 cells, expressing histone H2-GFP, to proceed faster through mitosis as compared to lymphoblastic DG-75 cells. While DG-75 cells exhibited a stable metaphase arrest upon spindle disruption using the spindle poison nocodazole, Kasumi-1 cells showed only a transient arrest, degraded securin and cyclin B and underwent sister-chromatid separation in the absence of a functional mitotic spindle. These findings suggest that the mitotic checkpoint is unable to properly interfere with APC/C-dependent proteolysis to prevent mitotic progression in myeloblastic leukemia upon treatment with spindle poisons. By using inducible retroviral reexpression of BubR1 and its downstream effector cyclin B we could enhance the ability of Kasumi-1 cells to accumulate in mitosis upon spindle disruption. Moreover, restoration of BubR1 led to higher cyclin B levels. Live-cell imaging analyses of Kasumi-1 cells, which expressed doxycyclin-inducible BubR1, revealed a prolonged metaphase, suggesting a more stringent control by the mitotic checkpoint when BubR1 expression is restored. Prolonged metaphase delays were also detected after reexpression of BubR1 when we challenged the cells with lower doses of spindle poisons suggesting that BubR1 is an important sensitizer for antimitotic therapies. Therefore, our finding of low BubR1 expression in AML provides an explanation for the poor response of myeloid leukemia to spindle poisons as compared to lymphoblastic leukemia. BubR1 has also been reported to be inactivated through promoter hypermethylation in various malignancies. The existence of a CpG island in the upstream region of the Bub1b locus (BubR1 coding sequence) tempted us to treat Kasumi-1 cells with the demethylating agent decitabine. Promotor demethylation led to an upregulation of BubR1 in mitotic AML cells providing evidence that BubR1 is a druggable target to enhance mitotic surveillance in AML cells. Conclusions: Because mitotic therapies are widely used in the treatment of different malignancies, a further understanding of these processes might lead to a better understanding of cancer biology and improved therapeutic approaches. Disclosures: No relevant conflicts of interest to declare.

scholarly journals APC and EB1 Function Together in Mitosis to Regulate Spindle Dynamics and Chromosome Alignment

2005 ◽  
Vol 16 (10) ◽  
pp. 4609-4622 ◽  
Author(s):  
Rebecca A. Green ◽  
Roy Wollman ◽  
Kenneth B. Kaplan
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Functional Relationship ◽  
Microtubule Dynamics ◽  
Dominant Negative ◽  
Mitotic Spindles ◽  
Spindle Dynamics ◽  
Chromosome Alignment ◽  
Spindle Function ◽  
Insight Into

Recently, we have shown that a cancer causing truncation in adenomatous polyposis coli (APC) (APC1–1450) dominantly interferes with mitotic spindle function, suggesting APC regulates microtubule dynamics during mitosis. Here, we examine the possibility that APC mutants interfere with the function of EB1, a plus-end microtubule-binding protein that interacts with APC and is required for normal microtubule dynamics. We show that siRNA-mediated inhibition of APC, EB1, or APC and EB1 together give rise to similar defects in mitotic spindles and chromosome alignment without arresting cells in mitosis; in contrast inhibition of CLIP170 or LIS1 cause distinct spindle defects and mitotic arrest. We show that APC1–1450 acts as a dominant negative by forming a hetero-oligomer with the full-length APC and preventing it from interacting with EB1, which is consistent with a functional relationship between APC and EB1. Live-imaging of mitotic cells expressing EB1-GFP demonstrates that APC1–1450 compromises the dynamics of EB1-comets, increasing the frequency of EB1-GFP pausing. Together these data provide novel insight into how APC may regulate mitotic spindle function and how errors in chromosome segregation are tolerated in tumor cells.

scholarly journals Neuropeptide Y inhibits cholangiocarcinoma cell growth and invasion

2011 ◽  
Vol 300 (5) ◽  
pp. C1078-C1089 ◽  
Author(s):  
Sharon DeMorrow ◽  
Paolo Onori ◽  
Julie Venter ◽  
Pietro Invernizzi ◽  
Gabriel Frampton ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Growth ◽  
Tumor Cell ◽  
Neuropeptide Y ◽  
Cell Lines ◽  
Xenograft Model ◽  
Human Tumor ◽  
Cholangiocarcinoma Cell

No information exists on the role of neuropeptide Y (NPY) in cholangiocarcinoma growth. Therefore, we evaluated the expression and secretion of NPY and its subsequent effects on cholangiocarcinoma growth and invasion. Cholangiocarcinoma cell lines and nonmalignant cholangiocytes were used to assess NPY mRNA expression and protein secretion. NPY expression was assessed by immunohistochemistry in human liver biopsies. Cell proliferation and migration were evaluated in vitro by MTS assays and matrigel invasion chambers, respectively, after treatment with NPY or a neutralizing NPY antibody. The effect of NPY or NPY depletion on tumor growth was assessed in vivo after treatment with NPY or the neutralizing NPY antibody in a xenograft model of cholangiocarcinoma. NPY secretion was upregulated in cholangiocarcinoma compared with normal cholangiocytes. Administration of exogenous NPY decreased proliferation and cell invasion in all cholangiocarcinoma cell lines studied and reduced tumor cell growth in vivo. In vitro, the effects of NPY on proliferation were blocked by specific inhibitors for NPY receptor Y2, but not Y1 or Y5, and were associated with an increase in intracellular d- myo-inositol 1,4,5-trisphosphate and PKCα activation. Blocking of NPY activity using a neutralizing antibody promoted cholangiocarcinoma growth in vitro and in vivo and increased the invasiveness of cholangiocarcinoma in vitro. Increased NPY immunoreactivity in human tumor tissue occurred predominantly in the center of the tumor, with less expression toward the invasion front of the tumor. We demonstrated that NPY expression is upregulated in cholangiocarcinoma, which exerts local control on tumor cell proliferation and invasion. Modulation of NPY secretion may be important for the management of cholangiocarcinoma.

Novel Symmetrical 1,4-Disubstituted-bis-1,2,3-Triazoles: Synthesis by Double CuAAC and Cytotoxicity Evaluation

2018 ◽  
Vol 18 (17) ◽  
pp. 1475-1482 ◽  
Author(s):  
Wallace J. Reis ◽  
Paulo O.L. Moreira ◽  
Rosemeire B. Alves ◽  
Heloísa H.M. Oliveira ◽  
Luciana M. Silva ◽  
...  
Keyword(s):  
Cell Lines ◽  
Human Tumor ◽  
Breast Adenocarcinoma ◽  
Tetraethylene Glycol ◽  
Ovarian Adenocarcinoma ◽  
Standard Drug ◽  
Human Tumor Cell Lines ◽  
Induced Apoptosis ◽  
Brdu Assay

Background: A series of symmetrical 1,4-disubstituted bis-1,2,3-triazoles was prepared by double copper catalyzed Azide-alkyne Cycloaddition (CuAAC) from aliphatic bis-azides and a tetraethylene glycol bis-azide derivative. The eighteen novel compounds were evaluated in vitro for their cytotoxic activity against two human tumor cell lines: Human breast adenocarcinoma (MDA-MB 231) and ovarian adenocarcinoma (TOV-21G). Results and Conclusion: The results of colorimetric MTT assays showed that compounds 4j and 4q exhibited a better selectivity index and cell viability comparable with the standard drug doxorubicin. These compounds induced apoptosis in both tested cell lines, as assessed by BrdU assay. The results suggest that these structurally simple compounds may be promising prototypes for antitumoral agents.

scholarly journals Involvement of PPARs in Cell Proliferation and Apoptosis in Human Colon Cancer Specimens and in Normal and Cancer Cell Lines

PPAR Research ◽  
2007 ◽  
Vol 2007 ◽  
pp. 1-9 ◽  
Author(s):  
G. Martinasso ◽  
M. Oraldi ◽  
A. Trombetta ◽  
M. Maggiora ◽  
O. Bertetto ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Cell Proliferation ◽  
Cell Lines ◽  
Human Tumor ◽  
Inverse Correlation ◽  
Human Colon ◽  
Human Colon Cancer ◽  
Ppar Ligands

PPAR involvement in cell growth was investigated “in vivo” and “in vitro” and was correlated with cell proliferation and apoptotic death. “In vivo” PPARγandαwere evaluated in colon cancer specimens and adjacent nonneoplastic colonic mucosa. PPARγincreased in most cancer specimens versus mucosa, with a decrease in c-Myc and in PCNA proteins, suggesting that colon cancer growth is due to increased cell survival rather than increased proliferation. The prevalence of survival over proliferation was confirmed by Bcl-2 or Bcl-XLincrease in cancer versus mucosa, and by decreased PPARα. “In vitro” PPARγand PPARαwere evaluated in human tumor and normal cell lines, treated with natural or synthetic ligands. PPARγwas involved in inhibiting cell proliferation with a decrease in c-Myc protein, whereas PPARαwas involved in inducing apoptosis with modulation of Bcl-2 and Bad proteins. This involvement was confirmed using specific antagonists of two PPARs. Moreover, the results obtained on treating cell lines with PPAR ligands confirm observations in colon cancer: there is an inverse correlation between PPARαand Bcl-2 and between PPARγand c-Myc.

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