Comparison of the in vitro effects of colchicine and its derivative colchiceine on chondrocyte morphology and function

Summary Colchiceine is a colchicine-metabolite which has been reported to inhibit axonal transport although not binding to brain tubulin. In the present study, colchiceine was shown not to depolymerize cytoplasmic microtubules, nor to mimic other effects of colchicine on the ultrastructure of cultured chondrocytes. In addition, the synthesis of proteoglycans was inhibited by colchicine but slightly stimulated by colchiceine. These results support the idea that the disturbances in cultured chondrocytes caused by colchicine are specifically related to a loss of cytoplasmic microtubules.This work was supported by grants from the Swedish Medical Research Council (proj. No. 12X-03355), the King Gustaf V 80th Birthday Fund, the Swedish Society of Medical Sciences, and from the funds of Karolinska Institutet.     

Is tubulin involved in the electrically-induced mechanical activity of the isolated rat sciatic nerve?

Summary The electrically-induced mechanical activity of isolated segments of rat sciatic nerves remains unaffected following incubation with 10^–4 M colchicine, vinblastine or melatonin. Vinblastine depressed tubulin levels in incubated nerves. These results suggest that microtubules are not involved in nerve mechanical activity in vitro.This work was supported by grant No. 6638 from the CONICET (Argentina). It is dedicated to the memory of C.E. Valenti, decreased November 1979.     

Coating of centrospheres by reticulum cisternae and formation of spindle residues in segmentation mitosis under the action of an antitubulin substance: nocodazole]

When cleaving eggs are treated by nocodazole the reticulum cisternae coat the centrospheres instead of the chromosomes, as normally occurs at the late anaphase. At the same time the spindle is reduced to a common mass or spindle remnant, constituted of unorientated and fragmented microtubules which embed the kinetochores, i. e. the star configuration of the chromosomes. This action is quite similar to that of chloralhydrate but is different from that of colchicine.     

Role of lysosomes, microtubules and microfilaments in the mechanism of the lactogenic action of prolactin in the rabbit mammary gland]

The lysosomotropic agents, ammonium chloride and chloroquine, added to the culture medium of pseudopregnant Rabbit mammary gland, did not inhibit the initiation of casein synthesis by prolactin. By contrast, they considerably reduced the down-regulation of prolacting receptors. Converse-y, colchicine totally blocked the lactogenic action of prolactin without altering the down-regulation of the receptor. Cytochalasin B inhibited only partly the lactogenic action of prolactin while it has no effect on the down-regulation of the receptor. These data suggest that the degradation of the prolactin-receptor complex in lysosome is not a compulsory step in the mechanism of prolactin action. The integrity of microtubules but not of microfilaments is required for prolactin to initiate casein synthesis. These elements of the cytoskeleton are not strictly involved in the down-regulation of the receptor.     

Transport and diffusion of Tau protein in neurons

In highly polarized and elongated cells such as neurons, Tau protein must enter and move down the axon to fulfill its biological task of stabilizing axonal microtubules. Therefore, cellular systems for distributing Tau molecules are needed. This review discusses different mechanisms that have been proposed to contribute to the dispersion of Tau molecules in neurons. They include (1) directed transport along microtubules as cargo of tubulin complexes and/or motor proteins, (2) diffusion, either through the cytosolic space or along microtubules, and (3) mRNA-based mechanisms such as transport of Tau mRNA into axons and local translation. Diffusion along the microtubule lattice or through the cytosol appear to be the major mechanisms for axonal distribution of Tau protein in the short-to-intermediate range over distances of up to a millimetre. The high diffusion coefficients ensure that Tau can distribute evenly throughout the axonal volume as well as along microtubules. Motor protein-dependent transport of Tau dominates over longer distances and time scales. At low near-physiological levels, Tau is co-transported along with short microtubules from cell bodies into axons by cytoplasmic dynein and kinesin family members at rates of slow axonal transport.     

Microtubules, interkinetic nuclear migration and neurulation.

The hypotheses dealing with mechanisms of neurulation are reviewed briefly. The phenomenon of interkinetic nuclear migration is thought to be an important factor to be considered in the invagination of the neuroepithelium in the chick embryo. Evidence is presented that implicates cytoplasmic microtubules in this phenomenon. It is suggested that microtubules not only participate in cell elongation but also that they are involved, through interkinetic nuclear migration, in the broadening of the basal region of the cells; this widening progressively creates the strain that ensures the invagination of the neuroepithelium.     

Action of diphenylhydantoin on chromosomes

The effect of diphenyl hydantoin, (DPH) a nonbarbituate anticonvulsant drug, on chromosomes and fertility was tested in cultured human lymphocytes, mouse fertility, and rat maternal marrow chromosomes and fetal development. Whole human blood from 5 male and 5 female subjects was cultured for 68 hours with phytohemagglutinin, then incubated for 2 hours in isotonic salts with .05-.3 mg per ml DPH, .02 mcg per ml colchicine, or .4 mg per ml sodium diethylbarbiturate. The mean number of metaphases per 1000 stimulated cells was 10.0 in controls, 40.3 with colchicine, 27.9 with diethylbarbiturate, and 30.5 with .25 mg DPH per ml. Both diphenylbarbiturate and DPH produced linear dose effect curves. These results were demonstrated not to be due to urea, since there were no differences in urea content, with a 2 hour urease micromethod. Mouse fertility was totally inhibited in 6 virgin mice given .1mg DPH daily for 10 days compared to 41 pups both of 6 control mice. In 6 pregnant rats given 25 mg DPH per 100 gm/orally 4 times daily for 2 days on gestation Days 7 and 8, there were 5 rats with all fetuses resorbed and 1 rat with 3 living and several resorptions. 6 controls had 6-14 normal fetuses each. 50 metaphase plates from each rat's femoral marrow and each fetus were examined 2 hous after injecting .3 mg colchicine per 100 gm. 30% of the metaphase cells from treated females and fetuses showed strongly contracted chromosomes and reduced number os "pulverized" chromosomes. These phenomena may have been due to inhibition by DPH of folic acid metabolism which is involved in purine synthesis.     

Microtubules,interkinetic nuclear migration and neurulation

Summary The hypotheses dealing with mechanisms of neurulation are reviewed briefly. The phenomenon of interkinetic nuclear migration is thought to be an important factor to be considered in the invagination of the neuroepithelium in the chick embryo. Evidence is presented that implicates cytoplasmic microtubules in this phenomenon. It is suggested that microtubules not only participate in cell elongation but also that they are involved, through interkinetic nuclear migration, in the broadening of the basal region of the cells; this widening progressively creates the strain that ensures the invagination of the neuroepithelium.Acknowledgments. This work is supported by the MRC of Canada. We are grateful to C. Auclair for his invaluable collaboration and to Miss H. Stephens for her help in improving the English composition of this text.     

Impaired nuclear translocation of glucocorticoid receptors: novel findings from psoriatic epidermal keratinocytes

Psoriasis is a chronic proliferative skin disease and is usually treated with topical glucocorticoids, which act through the glucocorticoid receptor (GR), a component of the physiological systems essential for immune responses, differentiation, and homeostasis. To investigate the possible role of GR in the pathogenesis of psoriasis, normal and psoriatic lesional skin were recruited. Firstly, the immunolocalization of GR in the skin and cultured epidermal keratinocytes were determined by immunofluorescence. In normal skin and cultured human epidermal keratinocytes, intracellular GR is localized in the nuclei, while in psoriatic skin and cultured keratinocytes, GR is in the cytoplasm. Next, we investigated possible factors associated with the cytoplasmic distribution. We found that VEGF and IFN-γ led to impaired nuclear translocation of GR through p53 and microtubule-inhibitor, vincristine, and inhibited nuclear uptake of GR in normal keratinocytes. In addition to dexamethasone, interleukin (IL)-13 was also able to transfer GR into nuclei of psoriatic keratinocytes. Furthermore, discontinuation of dexamethasone induced cytoplasmic retention of GR in normal keratinocytes. In contrast, energy depletion of normal epidermal keratinocytes did not change the nuclear distribution of GR. To confirm our findings in vivo, an imiquimod-induced psoriasis-like skin mouse model was included. IL-13 ameliorated (but vincristine exacerbated) the skin lesions on the mouse. Taken together, our findings define that impaired nuclear translocation of GR is associated with VEGF, IFN-γ, p53, and microtubule. Therapeutic strategies designed to accumulate GR in the nucleus, such as IL-13, may be beneficial for the therapy of psoriasis.     

Hexosaminidase and alkaline phosphatase activities in articular chondrocytes and relationship to cell culture conditions

Hexosaminidase and alkaline phosphatase activities in rabbit articular chondrocytes have been studied under different cell culture conditions. Chondrocytes were cultured in monolayer primary culture, monolayer subcultured to the fifth passage (in vitro aging) and cultured within a collagen gel; enzymatically released cartilage cells were used as control. Under these conditions, the two enzymes behave quite differently in relationship to alteration of the chondrocyte phenotype in culture. Increased lysosomal hexosaminidase activity could be considered to be a marker of the dedifferentiated phenotype in monolayer subculture; membrane alkaline phosphatase activity could be used as a marker of non-proliferating cells.     

Hexosaminidase and alkaline phosphatase activities in articular chondrocytes and relationship to cell culture conditions.

Hexosaminidase and alkaline phosphatase activities in rabbit articular chondrocytes have been studied under different cell culture conditions. Chondrocytes were cultured in monolayer primary culture, monolayer subcultured to the fifth passage (in vitro aging) and cultured within a collagen gel; enzymatically released cartilage cells were used as control. Under these conditions, the two enzymes behave quite differently in relationship to alteration of the chondrocyte phenotype in culture. Increased lysosomal hexosaminidase activity could be considered to be a marker of the dedifferentiated phenotype in monolayer subculture; membrane alkaline phosphatase activity could be used as a marker of non-proliferating cells.     

Colchicine inhibition of ADH effect on frog skin permeability

ADH and AMPc enhance both thiourea unidirectional fluxes in frog skin. This effect is completely abolished by colchicine pretreatment. The ADH increase of thiourea discharge with or without colchicine led us to suppose that colchicine does not directly affect ADH action on outer membrane permeability, but exerts its effects on a site which is limiting for the ADH action on transepithelial permeability.     

Effect of colchicine on polymerization of tubulin from rats,mice, hamsters and guinea-pigs

Summary Colchicine-inhibition of polymerization of tubulin from rats, mice, golden hamsters and guinea-pigs was studied to determine if species differences in tubulin sensitivity to colchicine might parallel species variation in colchicine toxicity. It was found that polymerization of tubulin is nearly equally sensitive to colchicine in all four species.This work was supported by PHS Grant No. CA 16425.     

Colchicine-induced appearance (proliferation) of smooth sarcoplasmic reticulum in arterial smooth muscle cells

Colchicine treatment resulted in the appearance and proliferation of smooth sarcoplasmic reticulum in some smooth muscle cells of the aortic and pulmonary trunk walls in the rabbit. The significance of cytoplasmic microtubules and/or membrane-bound tubulin for the morphogenesis, functioning and control of smooth endoplasmic reticulum in different kinds of cells is discussed.     

Colchicine-induced appearance (proliferation) of smooth sarcoplasmic reticulum in arterial smooth muscle cells

Summary Colchicine treatment resulted in the appearance and proliferation of smooth sarcoplasmic reticulum in some smooth muscle cells of the aortic and pulmonary trunk walls in the rabbit. The significance of cytoplasmic microtubules and/or membrane-bound tubulin for the morphogenesis, functioning and control of smooth endoplasmic reticulum in different kinds of cells is discussed.     

Colchicine inhibition of ADH effect on frog skin permeability

Summary ADH and AMPc enhance both thiourea unidirectional fluxes in frog skin. This effect is completely abolished by colchicine pretreatment. The ADH increase of thiourea discharge with or without colchicine led us to suppose that colchicine does not directly affect ADH action on outer membrane permeability, but exerts its effects on a site which is limiting for the ADH action on transepithelial permeability.The present work has been supported by C.N.R., Rome.     

Effect of colchicine on polymerization of tubulin from rats, mice, hamsters and guinea-pigs.

Colchicine-inhibition of polymerization of tulbulin from rats, mice, golden hamsters and guinea-pigs was studied to determine if species differences in tubulin sensitivity to colchicine might parallel species variation in colchicine toxicity. It was found that polymerization of tubulin is nearly equally sensitive to colchicine in all four species.     

The growth responses of hamster chondrocytes,dermal fibroblasts and embryo cells to whole blood serum and plasma-derived serum

Summary Autoradiographic studies with³H-thymidine demonstrated that the growth responses of hamster chondrocytes, dermal fibroblasts and embryo cells, respectively, differed in media containing whole blood serum (WBS) and plasmaderived serum (PDS). Dermal fibroblasts seemed to require a growth factor from platelets for growth, but chondrocytes did not. Embryo cells showed an intermediate pattern of growth response to this factor.This work was supported by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture and the Ministry of Health and Welfare of Japan.We thank Miss M. Tanaka and Miss K. Kawana for technical assistance.     

IFT54 regulates IFT20 stability but is not essential for tubulin transport during ciliogenesis

Intraflagellar transport (IFT) is required for ciliogenesis by ferrying ciliary components using IFT complexes as cargo adaptors. IFT54 is a component of the IFT-B complex and is also associated with cytoplasmic microtubules (MTs). Loss of IFT54 impairs cilia assembly as well as cytoplasmic MT dynamics. The N-terminal calponin homology (CH) domain of IFT54 interacts with tubulins/MTs and has been proposed to transport tubulin during ciliogenesis, whereas the C-terminal coiled-coil (CC) domain binds IFT20. However, the precise function of these domains in vivo is not well understood. We showed that in Chlamydomonas, loss of IFT54 completely blocks ciliogenesis but does not affect spindle formation and proper cell cycle progression, even though IFT54 interacts with mitotic MTs. Interestingly, IFT54 lacking the CH domain allows proper flagellar assembly. The CH domain is required for the association of IFT54 with the axoneme but not with mitotic MTs, and also regulates the flagellar import of IFT54 but not IFT81 and IFT46. The C-terminal CC domain is essential for IFT54 to bind IFT20, and for its recruitment to the basal body and incorporation into IFT complexes. Complete loss of IFT54 or the CC domain destabilizes IFT20. ift54 mutant cells expressing the CC domain alone rescue the stability of IFT20 and form stunted flagella with accumulation of both IFT-A component IFT43 and IFT-B component IFT46, indicating that IFT54 also functions in IFT turn-around at the flagellar tip.     

In situ aging of auricular chondrocytes is not due to the exhaustion of their replicative potential

Summary Multiplication of chondrocytes during growth of rabbit auricular cartilage was estimated on the basis of total DNA determination and compared with the population doubling level reached by these chondrocytes in vitro. The results indicate that the in situ aging of auricular chondrocytes is caused by factors other than the intrinsic depletion of their growth potential.