Irina Matos

p63, cytokeratin 5, and P-cadherin: three molecular markers to distinguish basal phenotype in breast carcinomas

Human breast carcinomas represent a heterogeneous group of tumors diverse in behavior, outcome, and response to therapy. However, the current system of pathological classification does not take into account biologic determinants of prognosis. The purpose of this study was to classify and characterize breast carcinomas based on variations in protein expression patterns derived from immunohistochemical analyses on tissue microarrays (TMAs). Therefore, 11 TMAs representing 168 invasive breast carcinomas were constructed. Breast tumors were classified into four different subtypes depending on estrogen receptor (ER) and HER2 expression. Basal-type tumors expressed neither of these proteins and represented 7.6% of our series; basal-like HER2-overexpressing tumors did not express ER and represented 17.7%; luminal-type tumors expressed ER and represented 72.8% of this series (luminal A 56.3%, luminal B 16.5%). Moreover, we characterized each subtype based on P-cadherin (P-CD), p63, cytokeratin (CK)5, BCL2, and Ki67 expression. Basal-type tumors were mostly grade III, more frequently P-CD-, p63-, and CK5-positive, and had a high proliferation rate. Conversely, luminal-type tumors rarely expressed basal markers and had a low grade and proliferation rate. Basal-like HER2-overexpressing tumors showed a basal-type profile similar with a high grade and up-regulation of P-CD and CK5. With this study, we show that P-CD, p63, and CK5 are important molecular markers that can be used to distinguish a basal phenotype. In addition, we also demonstrate the usefulness of TMAs in breast carcinoma immunoprofiling.

Synchronizing chromosome segregation by flux-dependent force equalization at kinetochores

The synchronous movement of chromosomes during anaphase ensures their correct inheritance in every cell division. This reflects the uniformity of spindle forces acting on chromosomes and their simultaneous entry into anaphase. Although anaphase onset is controlled by the spindle assembly checkpoint, it remains unknown how spindle forces are uniformly distributed among different chromosomes. In this paper, we show that tension uniformity at metaphase kinetochores and subsequent anaphase synchrony in Drosophila S2 cells are promoted by spindle microtubule flux. These results can be explained by a mechanical model of the spindle where microtubule poleward translocation events associated with flux reflect relaxation of the kinetochore–microtubule interface, which accounts for the redistribution and convergence of kinetochore tensions in a timescale comparable to typical metaphase duration. As predicted by the model, experimental acceleration of mitosis precludes tension equalization and anaphase synchrony. We propose that flux-dependent equalization of kinetochore tensions ensures a timely and uniform maturation of kinetochore–microtubule interfaces necessary for error-free and coordinated segregation of chromosomes in anaphase.

Feedback control of chromosome separation by a midzone Aurora B gradient

Taking a check on chromosome spacing Animal cells divide by mitosis. Chromosomes become condensed and congregate on the mitotic spindle in the center of the cell—the midzone. The spindle then separates sister chromosomes, pulling them to opposite ends of the cell, ready to form new daughter nuclei. Afonso et al. now show that chromosome separation is monitored by the level of midzone-associated Aurora B kinase activity (see the Perspective by Hadders and Lens). This process ensures that daughter nuclei only reassemble after sister chromosomes have successfully separated. Science, this issue p. 332; see also p. 265 A mitotic spindle midzone-associated Aurora B gradient monitors chromosome separation during cell division. [Also see Perspective by Hadders and Lens] Accurate chromosome segregation during mitosis requires the physical separation of sister chromatids before nuclear envelope reassembly (NER). However, how these two processes are coordinated remains unknown. Here, we identified a conserved feedback control mechanism that delays chromosome decondensation and NER in response to incomplete chromosome separation during anaphase. A midzone-associated Aurora B gradient was found to monitor chromosome position along the division axis and to prevent premature chromosome decondensation by retaining Condensin I. PP1/PP2A phosphatases counteracted this gradient and promoted chromosome decondensation and NER. Thus, an Aurora B gradient appears to mediate a surveillance mechanism that prevents chromosome decondensation and NER until effective separation of sister chromatids is achieved. This allows the correction and reintegration of lagging chromosomes in the main nuclei before completion of NER.

Dynein and mast/orbit/CLASP have antagonistic roles in regulating kinetochore-microtubule plus-end dynamics.

Establishment and maintenance of the mitotic spindle requires the balanced activity of microtubule-associated proteins and motors. In this study we have addressed how the microtubule plus-end tracking protein Mast/Orbit/CLASP and cytoplasmic dynein regulate this process in Drosophila melanogaster embryos and S2 cells. We show that Mast accumulates at kinetochores early in mitosis, which is followed by a poleward streaming upon microtubule attachment. This leads to a reduction of Mast levels at kinetochores during metaphase and anaphase that depends largely on the microtubule minus end-directed motor cytoplasmic dynein. Surprisingly, we also found that co-depletion of Dynein rescues spindle bipolarity in Mast-depleted cells, while restoring normal microtubule poleward flux. Our results suggest that Mast and Dynein have antagonistic roles in the local regulation of microtubule plus-end dynamics at kinetochores, which are important for the maintenance of spindle bipolarity and normal spindle length.

Dissecting mitosis with laser microsurgery and RNAi in Drosophila cells.

Progress from our present understanding of the mechanisms behind mitosis has been compromised by the fact that model systems that were ideal for molecular and genetic studies (such as yeasts, C. elegans, or Drosophila) were not suitable for intracellular micromanipulation. Unfortunately, those systems that were appropriate for micromanipulation (such as newt lung cells, PtK1 cells, or insect spermatocytes) are not amenable for molecular studies. We believe that we can significantly broaden this scenario by developing high-resolution live cell microscopy tools in a system where micromanipulation studies could be combined with modern gene-interference techniques. Here we describe a series of methodologies for the functional dissection of mitosis by the use of simultaneous live cell microscopy and state-of-the-art laser microsurgery, combined with RNA interference (RNAi) in Drosophila cell lines stably expressing fluorescent markers. This technological synergism allows the specific targeting and manipulation of several structural components of the mitotic apparatus in different genetic backgrounds, at the highest spatial and temporal resolution. Finally, we demonstrate the successful adaptation of agar overlay flattening techniques to human HeLa cells and discuss the advantages of its use for laser micromanipulation and molecular studies of mitosis in mammals.

A chromosome separation checkpoint

Here we discuss a “chromosome separation checkpoint” that might regulate the anaphase‐telophase transition. The concept of cell cycle checkpoints was originally proposed to account for extrinsic control mechanisms that ensure the order of cell cycle events. Several checkpoints have been shown to regulate major cell cycle transitions, namely at G1‐S and G2‐M. At the onset of mitosis, the prophase‐prometaphase transition is controlled by several potential checkpoints, including the antephase checkpoint, while the spindle assembly checkpoint guards the metaphase‐anaphase transition. Our hypothesis is based on the recently uncovered feedback control mechanism that delays chromosome decondensation and nuclear envelope reassembly until effective separation of sister chromatids during anaphase is achieved. A central player in this potential checkpoint is the establishment of a constitutive, midzone‐based Aurora B phosphorylation gradient that monitors the position of chromosomes along the spindle axis. We propose that this surveillance mechanism represents an additional step towards ensuring mitotic fidelity.

Drosophila S2 cells as a model system to investigate mitotic spindle dynamics, architecture, and function

In order to perpetuate their genetic content, eukaryotic cells have developed a microtubule-based machine known as the mitotic spindle. Independently of the system studied, mitotic spindles share at least one common characteristic--the dynamic nature of microtubules. This property allows the constant plasticity needed to assemble a bipolar structure, make proper kinetochore-microtubule attachments, segregate chromosomes, and finally disassemble the spindle and reform an interphase microtubule array. Here, we describe a variety of experimental approaches currently used in our laboratory to study microtubule dynamics during mitosis using Drosophila melanogaster S2 cells as a model. By using quantitative live cell imaging microscopy in combination with an advantageous labeling background, we illustrate how several cooperative pathways are used to build functional mitotic spindles. We illustrate different ways of perturbing spindle microtubule dynamics, including pharmacological inhibition and RNA interference of proteins that directly or indirectly impair microtubule dynamics. Additionally, we demonstrate the advantage of using fluorescent speckle microscopy to investigate an intrinsic property of spindle microtubules known as poleward flux. Finally, we developed a set of laser microsurgery-based experiments that allow, with unique spatiotemporal resolution, the study of specific spindle structures (e.g., centrosomes, microtubules, and kinetochores) and their respective roles during mitosis.

Tissue microarrays for testing basal biomarkers in familial breast cancer cases

CONTEXT AND OBJECTIVE The proteins p63, p-cadherin and CK5 are consistently expressed by the basal and myoepithelial cells of the breast, although their expression in sporadic and familial breast cancer cases has yet to be fully defined. The aim here was to study the basal immunoprofile of a breast cancer case series using tissue microarray technology. DESIGN AND SETTING This was a cross-sectional study at Universidade Estadual de Campinas, Brazil, and the Institute of Pathology and Molecular Immunology, Porto, Portugal. METHODS Immunohistochemistry using the antibodies p63, CK5 and p-cadherin, and also estrogen receptor (ER) and Human Epidermal Receptor Growth Factor 2 (HER2), was per-formed on 168 samples from a breast cancer case series. The criteria for identifying women at high risk were based on those of the Breast Cancer Linkage Consortium. RESULTS Familial tumors were more frequently positive for the p-cadherin (p = 0.0004), p63 (p < 0.0001) and CK5 (p < 0.0001) than was sporadic cancer. Moreover, familial tumors had coexpression of the basal biomarkers CK5+/ p63+, grouped two by two (OR = 34.34), while absence of coexpression (OR = 0.13) was associated with the sporadic cancer phenotype. CONCLUSION Familial breast cancer was found to be associated with basal biomarkers, using tissue microarray technology. Therefore, characterization of the familial breast cancer phenotype will improve the understanding of breast carcinogenesis.

Spatial control of the anaphase-telophase transition

The ultimate goal of mitosis is to physically separate the entire set of duplicated chromosomes in order to propagate the genetic information during cell division. The perpetuation of a faithful maintenance of the genome integrity can only be guaranteed by the existence of multiple cell cycle checkpoints.1 In particular, mitosis is a highly regulated process where checkpoints assume a major importance.2 The latest stages of mitosis have been extensively studied; however, the mechanisms underlying the coordination between chromosome separation during anaphase and mitotic exit remain to be unveiled. It has long been assumed that anaphase and telophase are events that occur passively after satisfaction of the spindle-assembly checkpoint (SAC).2 However, it seems counterintuitive to assume that these crucial moments of mitosis are not being tightly monitored. A basic assumption for successful chromosome segregation during anaphase is that the 2 sets of chromosomes effectively separate before nuclear envelope reformation (NER). Recently, we showed that an inverse correlation exists between chromosome separation velocity and anaphase duration in Drosophila and human cultured cells, suggesting the presence of an additional surveillance mechanism that spatially regulates the anaphase-telophase transition.3 Importantly, spatial regulation implies the presence of a sensory ruler capable of “measuring” the chromosome position along the division axis. In fact, we showed that the previously reported Aurora B phosphorylation gradient4 creates an “area of exclusion” that inhibits DNA decondensation and NER (Fig. 1). When global Aurora B activity is impaired, or its localization at the spindle midzone affected, this spatial regulation is lost and NER occurs independently of the chromosome position. The precocious assembly of the nuclear envelope around unseparated chromosomes may lead to aneuploidy and/or polyploidy that will ultimately perturb cell or tissue homeostasis. Figure 1. After SAC satisfaction, mitotic cells proceed into anaphase. Aurora B is transferred to the spindle midzone and establishes a phosphorylation gradient where its substrates are phosphorylated and chromosomes remain condensed. This phosphorylation gradient ... Although Aurora B activity dictates a spatial control of the anaphase-telophase transition, the prolonged extension of anaphase after the expression of non-degradable Cyclin B1 mutants indicates that a final step of degradation is required to determine true mitotic exit. Additionally, inhibition of Cdk1 activity during anaphase shortens anaphase duration, suggesting that Cyclin B1 is necessary to provide time for proper chromosome separation. Therefore, both Aurora B and Cdk1 kinase activities must be coordinated to assure the spatiotemporal control of the anaphase-telophase transition. An additional level of regulation is related to the phosphatases, which must be in place to counteract the kinase activities. Inhibition of PP1/PP2A at the anaphase onset caused an “endless anaphase” phenotype, with persistent condensed chromosomes and no evident NER. Our data suggests that the kinase activity is predominant in the spindle midzone and, as chromosomes move to opposite poles, phosphatase activity will prevail and account for the dephosphorylation of substrates and consequent mitotic exit (Fig. 1). Some outstanding questions still remain and in the future, it would be important to understand how AuroraB, Cdk1 and PP1/PP2A activities are mechanistically coordinated: is there a feedback loop combining these 3 players? Or is there a hierarchical series of phosphorylation/dephosphorylation events that promote the balanced activity that ultimately leads to accurate chromosome segregation and NER? In the context of a potential chromosome separation checkpoint it will be important to understand which are the substrates that mediate the anaphase-telophase transition in coordination with chromosome separation. In this context, the localization of the Drosophila Condensin I subunit Barren, whose loading onto chromosomes is dependent on Aurora B,5 was found to be sensitive to the midzone Aurora B phosphorylation gradient (Fig. 1). Additionally, Barren depletion caused a similar phenotype to the one found with Aurora B inhibition, further supporting that chromosome condensation could be a readout for chromosome position during anaphase. Overall, sister-chromatid separation during anaphase is actively monitored by the presence of a phosphorylation gradient of Aurora B established at the spindle midzone. This hub of kinase activity constitutively phosphorylates its substrates as long as they are under the influence of the gradient. The inhibitory action of Aurora B activity must therefore feedback to Cdk1 in order to dampen the kinase activity and allow the phosphatases to prevail. The proposed feedback control of the anaphase-telophase transition is just the tip of the iceberg - how it is molecularly regulated and whether this mechanism is conserved between different cells in different tissues and organisms will certainly be a direction of further research.