Enhancement of the transformed shape phenotype by microtubule inhibitors and reversal by an inhibitor combination.

Heckman, Carol A.; Plummer, Howard K.; Mukherjee, Rama

doi:10.3892/ijo.16.4.709

Cited by 6 publications

(38 citation statements)

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“…It was purchased from the LC Laboratories (Woburn, MA) and made up like paclitaxel. It was reported previously that substituting taxanes, baccatin III or cephalomannine, for paclitaxel did not lead to phenotype reversal (28). Therefore, paclitaxel was the only microtubule-stabilizing compound used in combination treatments.…”

Section: Methodsmentioning

confidence: 99%

“…All chemicals were from SigmaAldrich Company (St. Louis, MO) unless otherwise noted. Colchicine, nocodazole, podophyllotoxin, and vinblastine sulphate were made up and stored at -20˚C, as previously described (28). Paclitaxel (Taxol ® ) was obtained from the Development Therapeutics Program, Division of Cancer Treatment, National Cancer Institute and from Dabur, Inc., India.…”

Section: Methodsmentioning

confidence: 99%

“…N357 monoclonal antibody against ß-tubulin from Balb/C57 mouse (Amersham Biosciences, Piscataway, NJ) was diluted 1:600 for use. Secondary antibodies, FITC-conjugated goat anti-mouse (U.S. Biochemical Corp., Cleveland, OH) or Alexa 488-conjugated goat anti-mouse (Molecular Probes, Eugene, OR), were used as previously described (28). Representative cells were photographed on a Zeiss Axiophot microscope equipped with x63 and x100 Neofluar lenses, using Kodak Ektachrome film or else recorded with a Princeton Instruments RTE/CCD camera and IBM-PC running MetaMorph 4.6r5 software (Universal Imaging Corp., Buckinghamshire, UK).…”

Section: Methodsmentioning

confidence: 99%

“…Previous results showed that inhibitor combinations composed of paclitaxel and colchicine caused reversal of the transformed cell phenotype (28). The combination of paclitaxel with different depolymerizing agents was studied in the current experiments.…”

Section: Cancer-type Reversal By Inhibitor Combinationsmentioning

confidence: 99%

“…Reversal of the shape phenotype was reported using this combination on unsynchronized cell populations. Thus, the greatest fraction of the population was necessarily composed of cells in the G1, S, and G2 phases (24,28). These and other studies (7) gave clinicians a rationale to conduct tests of such inhibitor combinations.…”

Section: Introductionmentioning

confidence: 99%

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Pattern analysis of microtubule-polymerizing and -depolymerizing agent combinations as cancer chemotherapies

Uppal

Li²,

Wendt³

et al. 2007

Int J Oncol

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Abstract. Subcellular distribution of mass can be analyzed by a technique that involves culturing cells on interferometers and digitizing their interference contours. Contour sampling resulted in 102 variables per cell, which were predictors of oncogenic transformation. Cell phenotypes can be deconstructed by use of latent factors, which represent the covariance of the real variables. The reversal of the cancertype phenotype by a combination of microtubule-stabilizing and -depolymerizing agents was described previously. The implications of these results have been explored by clinicians who treated patients with the combination of docetaxel and vinorelbine (Navelbine ® ). The current study was performed to determine the effects of different combinations on phenotype and in phases of the cell cycle other than mitosis. Combinations of paclitaxel with either colchicine, podophyllotoxin, nocodazole, or vinblastine caused phenotype reversal. Paclitaxel analogue, 7-deoxytaxol, by itself caused reversal. Factors #4, (filopodia), #5 (displacement and/or deep invaginations in the periphery), #8, and #12 took on values typical of normal cells, whereas the values of #7 (p21-activated kinase), and #13 (rounding up) shifted toward the cancer-type. All combinations altered microtubule arrangement at the cell edge. Delivery schedules and drug ratios used in clinical studies were subjected to analysis. Clinical response rates were better when the combination was not interspersed with a single agent (P=0.004). The results support the idea that efficacy depends upon simultaneous exposure to both agents, and suggest a novel mechanism for combination therapies. These therapies appear to restore in transformed cells some of the features of a contactinhibited cell, and to impede progress through the cell cycle even when provided at nanomolar concentrations.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Cancer-type Reversal By Inhibitor Combinationsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Pattern analysis of microtubule-polymerizing and -depolymerizing agent combinations as cancer chemotherapies

Uppal

Li²,

Wendt³

et al. 2007

Int J Oncol

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Relationship of p21‐activated kinase (PAK) and filopodia to persistence and oncogenic transformation

Heckman

Demuth

Deters

et al. 2009

Journal Cellular Physiology

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Previously, we found that oncogenically transformed cells had fewer filopodia and more large, p21-activated kinase (PAK)-dependent features than normal cells. These large protrusions (LPs) were increased in cells expressing RhoA(N19) with Cdc42-associated kinase (ACK). Here, we determine how GTPase-mediated mechanisms of focal contact (FC) regulation affect these protrusions. Constructs encoding various proteins were introduced into cells which were then studied by microscopy and computerized image processing and analysis. Constructs that prevented PAK recruitment by PAK-interacting exchange factor (PIX) or restricted PAK residence time on FCs decreased both protrusions. Thus, filopodia were also PAK-dependent. A comparison of FC distribution in cells expressing PAK in the presence or absence of PAK kinase inhibitor domain (KID) suggested that PAK enlarged FCs without affecting the prevalence of either protrusion. KID or Nck expression increased LPs but not filopodia. Nck failed to synergize with KID or ACK and RhoA(N19) in enhancing LPs. Nck and KID synergistically enhanced filopodia, possibly because Nck recruited PAK to FCs while KID prevented their dissociation by PAK-mediated autophosphorylation. Coexpression of Nck, ACK, and RhoA(N19) abrogated filopodia and replicated the transformed phenotype. Since Nck recruitment of PAK is implicated in persistence of directional movement, we studied the PAK-Nck interface. Filopodia were eliminated by the Nck PAK-binding domain and LPs by the PAK Nck-binding domain. The results suggested that filopodia formation has more stringent requirements than LP formation, and Nck and PAK are used differently in the protrusions. Loss of filopodia in transformed cells may reflect defective regulation of GTPase mechanisms.

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Cell Edge Features Affected by Microtubule Inhibitor Combinations

et al. 2006

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The tumor promoter, phorbol 12-myristate 13-acetate (PMA), enhances tumor yield through an epigenetic mechanism. PMA, like another promoter, phosphatase inhibitor okadaic acid, works by maintaining proteins in a phosphorylated state. In order to identify chemicals with promoter and antipromoter effects, this laboratory has developed a standard curve of morphogenetic changes using data from precancerous cell lines that eventually became neoplastic. Using the curve as a basis of comparison, we defined the "signature" phenotype as that adopted when a cell line became neoplastic. The results of solving for signature type disclosed that the microtubule-depolymerizing compound, colchicine, had a promoter-like effect [2]. The opposite effect was found if cells were exposed to paclitaxel (Taxol®) and colchicine [3]. Such findings gave clinicians a rationale to test microtubule inhibitor combinations [4]. Therapy is only effective when agents are administered nearly simultaneously. In the current work, we study the effect of inhibitor combinations on cell features defined by computer-assisted microscopy and classification methods based on latent factors.When administered to cells, paclitaxel or cephalomannine alone enhanced factor #7, a descriptor of protrusions dependent upon p21-associated kinase [5]. The agents left factor #4, representing filopodia or microspikes, unaffected. Paclitaxel administered along with various microtubuledepolymerizing compounds elevated factor #7. 1:1 combinations of paclitaxel with colchicine, nocoazole, or vinblastine, all had the same effect. A 3:1 molar ratio of paclitaxel to podophyllotoxin or vinblastine was similar. Thus, protrusions were commonly enhanced by taxanes alone or in combination with depolymerizing agent. The prevalence of filopodia, determined by comparing factor #4 values with control, was only affected by an equimolar combination of paclitaxel and colchicine or 3:1 molar ratio of paclitaxel to podophyllotoxin. This endpoint was unaffected by taxane exposure but did respond to a high concentration of the paclitaxel analog, 7-deoxytaxol. Morphological correlates of the two types of cell features were sought by immunofluorescence localization of microtubules in cells fixed with -20˚ methanol. Control cells exhibited numerous microtubules arranged parallel to the cell edge (Fig. 1a). Only where cells made contact did microtubules typically appear to be perpendicular to the cell edge. In contrast, after 2 h exposure to the combination of paclitaxel with colchicine, microtubules were rearranged so that a part of the array extended straight out from the peripheral cytoplasm (Fig. 1b). This effect was observed after exposure to paclitaxel with podophyllotoxin and vinblastine (Fig. 2) as well. From these studies, it can be concluded that both the features represented by #4 and #7 can be influenced by taxanes and for #4, particularly by the combination of paclitaxel with microtubule-depolymerizing agents. References[1] Work supported in part by DOD grant DAMD17-01-1-0484[2] C.

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Enhancement of the transformed shape phenotype by microtubule inhibitors and reversal by an inhibitor combination.

Cited by 6 publications

References 0 publications

Pattern analysis of microtubule-polymerizing and -depolymerizing agent combinations as cancer chemotherapies

Pattern analysis of microtubule-polymerizing and -depolymerizing agent combinations as cancer chemotherapies

Relationship of p21‐activated kinase (PAK) and filopodia to persistence and oncogenic transformation

Cell Edge Features Affected by Microtubule Inhibitor Combinations

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