ABHA-sensitive cell lines have functional G2/M checkpoints. Asynchronously growing cultures (Con) or cultures treated with ICRF193 (ICRF) or nocodazole (Noco.) were harvested after 24 h and analyzed by FACS for cell cycle status. In each case, the drugs produced prominent G2/M accumulation with both tumor cell lines. Similar experiments were performed with the topoisomerase II poison etoposide, which produced results similar to those of the topoisomerase II poison ICRF193 (our unpublished results).
The loss of the histone deacetylase inhibitor-sensitive checkpoint results in the cells attempting to undergo mitosis in an inappropriate state, leading to noncongression of the condensed chromosomes and ultimately missegregation at cytokinesis. It is surprising that despite the chromosome noncongression observed in the ABHA-treated tumor cells, they do not block in mitosis because of the imposition of an anaphase spindle assembly checkpoint (Sorger et al., 1997) but appear to continue through into G1 phase. This phenotype is typical of mutations in BUB1, MAD2, and CENP-E, which are all kinetochore proteins involved in establishing the anaphase checkpoint arrest (Schaar et al., 1997; Taylor and McKeon, 1997; Waters et al., 1998). The spindle checkpoint function appears to be intact in the cell lines used in this study, because they all block in mitosis when treated with nocodazole, and this may indicate that ABHA affects the expression or function of components of the anaphase checkpoint pathway, e.g., the BUBs and MADs.
The aberrant mitosis is likely to be a trigger for death in the ABHA-treated tumor cells. The ABHA-induced arrest in the resistant cells is in G2 phase, premitotic, before the activation of cyclin A/cdk2 in early G2 phase and cyclin B/cdc2 at G2/M, and this arrest is clearly defective in the ABHA-sensitive cells. Reintroducing a G2 arrest using ICRF193 reduced the level of cell death after ABHA treatment, providing further support for the protective role of the G2 arrest. However, it is also clear that ABHA disrupts the mitotic spindle checkpoint in the ABHA-sensitive lines. The relationship between the defective G2 arrest and disruption of the later mitotic spindle checkpoint in these cells is unclear, although they may possibly be related. A precedent for a connection between the earlier G2 checkpoint and the later mitotic checkpoint does exist. The caffeine-induced bypass of etoposide-induced G2/M arrest results in disruption of normal chromatid disjunction during mitosis in mammalian cells and catastrophic fracturing of the chromosome by the mitotic spindle during mitosis (Lock et al., 1994). Examination of the mitotic figures in similar caffeine bypass experiments reveals a noncongression phenotype very similar to that observed with ABHA treatment of sensitive cells (our unpublished observations). Thus, it may be that dysfunction of the G2 checkpoint may also disrupt the later mitotic spindle checkpoint, ensuring that cells with significant DNA damage die.
What is the nature of the ABHA-sensitive G2 checkpoint, and how is it inactivated in the tumor cell lines One possibility is that ABHA alters the expression of a gene or genes involved in G2/M progression, which results in the G2 arrest in resistant cells, and the regulator of this transcriptional program is defective in the ABHA-sensitive cells. An alternative mechanism is a response to the dramatic increase in histone acetylation observed after ABHA treatment of both sensitive and resistant cells (Qiu et al., 1999). This mechanism would prevent cells entering mitosis with hyperacetylated chromatin, which may in turn affect centromere and kinetochore function and thereby disrupt the spindle checkpoint. There may be a defect in either the sensing or signaling mechanism in the tumor cells that results in the loss of the ABHA-sensitive checkpoint arrest. In yeast, TSA has been shown to cause chromosome loss and to disrupt the localization of Swi6p, normally localized to centromeres and involved in normal sister chromatid disjunction (Ekwall et al., 1997). This effect is related to hyperacetylation of centromeric histones and results in missegregation of chromosomes during mitosis. It has also been demonstrated that mutation of the amino-terminal lysine residues normally acetylated in histone H4 results in a G2/M arrest (Megee et al., 1995), and mutations in one of the chromatin remodeling complexes also produces a G2/M arrest and affects the chromatin structure around centromeres (Tsuchiya et al., 1998). Thus in lower eukaryotes a checkpoint mechanism exists that senses the acetylation state of the chromatin and centromere integrity, which consequently may disrupt normal kinetochore function. Considering the high degree of conservation of cell cycle mechanisms from yeast to human, it is likely that the ABHA-sensitive G2 checkpoint we have described here is related to the chromatin acetylation state-sensitive G2/M checkpoint in yeast.
We thank Dr. J.B. Rattner for the gift of the human kinetochore antiserum, Dr. A.M Creighton for the ICRF193, Dr. Nick Saunders (University of Queensland) for the SCC cell line, and Drs. Sherilyn Goldstone and Sandra Pavey for critical reading of the manuscript. This work was supported by grants from the National Health and Medical Research Council of Australia and the Queensland Cancer Fund. B.G.G. is an Australian Research Fellow.
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Currently, SIMOLD 2083 alloy is well known as material with excellent mechanical characteristics such as extreme erosion resistance and self-hardening in high temperature and friction, so it is extensively utilized in the molding industries for food field. For more specifics, this material can be hardened by itself in the operating process when the working environment has friction and high temperature, so it is very appropriate for injection molding. Another benefit of SIMOLD 2083 alloy is of the stainless steel group; it is also suitable for making the molding cavities and cores for plastic products used in the food area. Meanwhile, the fabrication process of SIMOLD 2083 alloy with complex 3D surfaces meets several struggles for achieving small surface roughness and high productivity because of detrimental machining conditions. Therefore, in order to obtain the beneficial surface quality as well as ensure machining productivity, the cutting parameters should be chosen in a suitable range and be optimized to gain the optimal milling conditions. There are various parameters such as the cutting parameters and chemical composition of workpiece and tool affecting the quality characteristics of the milled components. In addition, surface roughness is one of the most characteristics of the milling parts. In this research, so as to guarantee the quality and productivity of machining part with the complex milling surfaces, the surface roughness and milling time were considered. Consequently, so as to achieve better surface finish of a milled product and ensure the productivity, the cutting parameters were regarded for multiobjective optimization problem.
In this research, the milled component with complex milling surfaces should fulfill the following requirements: (i) the surface roughness (y1) should be as small as possible to assure the quality of the milling inclined surface and (ii) the small milling time (y2) to enhance the machining productivity. A combination method of the TM, the RSM, and the MWCA is grown for balance among them as well as advance of the superiority responses. The optimal issue for the milled component is expressed in the following form.
Secondly, the nine fabricated experimental models were fabricated according to proposed the number of experiments, as depicted in Figure 5. Later on, the surface roughness (y1) was assessed by a Mitutoyo SJ-210 surface roughness tester (Japan), as illustrated in Figure 6. Besides, in order to transfer inclined surface into straight surface for measuring surface roughness, the mounting fixture was designed and fabricated for fixing the milled component as well as conveying measuring surfaces with the average of three different measurements, in addition to the machining time (y2) and that recorded by CNC machine, respectively. Therefore, the experimental results are shown in Table 3.
The sensitivity analysis and ANOVA were implemented for determining the effects and crucial contributions of cutting factors on the surface roughness and milling time. The results of ANOVA analysis showed that the parameters that have significant influences on surface roughness were spindle velocity (42.42%), feed rate (29.40%), and cutting depth (6.59%), respectively. Meanwhile, the only parameter that has the most influence degree on milling time was feed rate (92.6%).
Moreover, the results illustrated that the errors among forecasted results and experimental validations for the roughness surface and milling time are 2.04% and 5.39%, congruently. The experimental affirmations were proximate to the forecasted results. Therefore, the predicted results are appropriate with the certifications. According to the aforementioned results, the hybrid method is a powerful method for solving the multiobjective optimization issue for the cutting factors.
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