Immunoelectron microscopic studies on centromere-kinetochore complexes detached from chromosomes

The centromere-kinetochore complexes of Chinese hamster ovary (CHO) cells were detached and separated from the condensed chromatin by treatment with hydroxyurea and caffeine. By labelling the complex for immunoelectron microscopy (immuno-EM) with a mixture of antibodies against centromere proteins (anti-CENP-A,-B, -C) in some cells, we could demonstrate complete detachment of the complexes. No remnants were left at the bulk of condensed chromatin in these cells. In some mitotic cells complex and chromatin were found side by side. It could be shown that the fine structure of the separated material of the complex differs significantly from that of the rest of chromatin. The complex consists of proteins and DNA. This leads us to suppose that the organization of chromatin in the centromere-kinetochore complex is different.     

Ultrastructural studies on the lung colonization by nonmetastatic rat tumor cells

The events during the settlement of BSp73AS (AS) tumor cells in the syngeneic rat lung are described. Although AS cells show highly invasive behavior in vitro, subcutaneous primary tumors grow solidly without detectable metastatic spreading. However, AS cells when applied to the syngeneic rats via the tail vein give rise to lung colonies which grow rapidly at the site of the cells' primary arrest in the capillaries. The colonization comprises formation of microemboli, penetration of the endothelium including the basal lamina, and invasion of the lung tissue. Within two weeks, large colonies develop, thereby compressing, invading, and destroying their surroundings without detectable preference in direction. This establishment of AS tumors in the lung reflects the high invasive potential of AS cells and displays many of the morphological features observed during the formation of colonies of metastatic cell lines. Thus, we conclude that a nonmetastatic tumor cell line, such as AS, may possess almost the whole set of properties necessary for successful metastasis.     

Sequence of centromere separation: kinetochore formation and DNA replication in dicentric chromosomes showing premature centromere separation in rat cerebral cells

A subpopulation of rat cerebral endothelial cells, designated B1, exhibits an array of multicentric chromosomes. Because of the formation of bridges at anaphase, this cell population produced new types of multicentrics at every cell division. These chromosomes showed kinetochore proteins at every centromeric site and all centromeric regions replicated their DNA at the end of the S phase, more or less simultaneously. A new subpopulation of cells, designated B2, obtained from the original sample frozen at Wayne State University displayed several dicentrics. In contrast to B1 these chromosomes exhibit premature centromere separation as reported for mouse and human cell lines. These B2 dicentrics show only one site of kinetochore protein deposition. The timing of DNA replication around the centric region of prematurely separating centromere is also changed similar to the earlier reported premature DNA synthesis for mouse dicentrics. These observations suggest a universality of relationship between premature centromere separation, a lack of kinetochore formation, and early replication of the centric/pericentric DNA associated with these centromeres. The cause of sudden change from activity to inactivity of these chromosomes, though interesting, is not clear.     

Adhesion and spreading characterization of a rat tumor cell system exhibiting different metastatic behavior

The rat tumor cell system BSp73AS/BSp73ASML was investigated for its structural aspects. The non-metastasizing line BSp73AS has less nuclear atypism than the metastasizing line BSp73ASML. Microvilli are scanty and variable in length and structure; in BSp73ASML they cover the cells densely, are short and homogeneous in size. In vitro adhesion and spreading tests show structurally flexible BSp73AS cells which flatten completely onto substrata. BSp73ASML cells remain spherical and develop only small attachment areas. In vitro aggregation of the BSp73AS cells leads to round, tightly packed aggregates, their outer cell layer is epitheloid BSp73ASML cells form clusters of mostly ball-shaped cells. Surprisingly, as judged from EM images, the intercellular junctions in BSp73ASML cell clusters are qualitatively the same as in BSp73AS cell aggregates. However, the extent of apposition between cells in BSp73AS aggregates is much higher than between cells in the BSp73ASML clusters. BSp73ASML cells lack the ability to vary their shape. This is the prominent difference revealed by these tests between the two cell lines. Hence, this fact could play an important role in their different metastatic behavior in vivo. We speculate that the ability to vary the cell shape is necessary for expansive growth in the host environment but not for the metastatic spread.     

Characteristic alterations of the peritoneum after carbon dioxide pneumoperitoneum

Objective: Any route of entry into the abdomen contributes to alterations of the intraperitoneal organs with different clinical consequences. Characteristic alterations of the peritoneum after CO₂ pneumoperitoneum used in laparoscopic surgery is examined. Methods: A CO₂ pneumoperitoneum with an intraperitoneal pressure of 6 mmHg was applied for 30 min in 32 nude mice. In the course of 4 days, the animals were killed and the peritoneal surface of the abdominal wall was studied by means of scanning electron microscopy. Results: Already 2 h after release of the pneumoperitoneum, mesothelial cells were bulging up. The intercellular clefts thereby increased in size, and the underlying basal lamina became visible. This reaction peaked after 12 h. Subsequently, peritoneal macrophages and lymphocytes filled all gaps, thereby recovering the basal lamina. Conclusion: The morphologic integrity of the peritoneum is temporarily disturbed by a CO₂ pneumoperitoneum. Received: 9 March 1998/Accepted: 24 July 1998     

Are proteasomes involved in the formation of the kinetochore?

Proteasomes catalyse the degradation of proteins responsible for the regulation of mitosis enabling the cell to complete cell division. We have studied the effect of an inhibitor of the chymotrypsin-like activity of the proteasome on the trilaminar structure of the kinetochore in HeLa cells. Whereas a role for the proteasome in the degeneration of the kinetochore was predicted, we found instead that the inhibitor strongly retarded kinetochore development. We observed different developmental stages of the kinetochore from the fibrous ball of a prekinetochore to the mature kinetochore in one cell. The data presented here support the proposition that proteasomes are involved in kinetochore formation.accepted for publication by H. C. MacgregorDedicated to the memory of Prof. Dr. Daniel Mazia, a fatherly friend.     

Enhanced Ratio of Signals Enables Digital Mutation Scanning for Rare Allele Detection

The use of droplet digital PCR (ddPCR) for low-level DNA mutation detection in cancer, prenatal diagnosis, and infectious diseases is growing rapidly. However, although ddPCR has been implemented successfully for detection of rare mutations at pre-determined positions, no ddPCR adaptation for mutation scanning exists. Yet, frequently, clinically relevant mutations reside on multiple sequence positions in tumor suppressor genes or complex hotspot mutations in oncogenes. Here, we describe a combination of coamplification at lower denaturation temperature PCR (COLD-PCR) with ddPCR that enables digital mutation scanning within approximately 50-bp sections of a target amplicon. Two FAM/HEX-labeled hydrolysis probes matching the wild-type sequence are used during ddPCR. The ratio of FAM/HEX-positive droplets is constant when wild-type amplicons are amplified but deviates when mutations anywhere under the FAM or HEX probes are present. To enhance the change in FAM/HEX ratio, we employed COLD-PCR cycling conditions that enrich mutation-containing amplicons anywhere on the sequence. We validated COLD-ddPCR on multiple mutations in TP53 and in EGFR using serial mutation dilutions and cell-free circulating DNA samples, and demonstrate detection down to approximately 0.2% to 1.2% mutation abundance. COLD-ddPCR enables a simple, rapid, and robust two-fluorophore detection method for the identification of multiple mutations during ddPCR and potentially can identify unknown DNA variants present in the target sequence.In the era of personalized medicine, mutation detection methods that target mutations known to influence therapy response or clinical outcome are of great interest. Although real-time PCR methodologies have been described and are widely used for detecting mutations in clinical samples,^1–4 interest in digital PCR⁵ is rising in view of the unique aspects of the technology and the emergence of commercial droplet digital PCR (ddPCR) platforms.^5–8 ddPCR has been implemented in a variety of fields such as cancer biomarker and viral load detection, fetal screening, or library quantification for next-generation sequencing.⁹ One of the most common ddPCR applications is in the detection of known DNA variants present within a large excess of wild-type DNA, for instance in DNA from heterogeneous samples that harbor subclonal populations of mutated tumor cells.¹⁰In ddPCR, the amplification reaction is compartmentalized into microscopic emulsion-based droplets containing at most a few target molecules per droplet. By segregating the interrogated sample, the effect of in-droplet target competition is reduced, which translates into increased assay discrimination and facile determination of wild-type versus mutant status.¹¹ However, as currently applied, ddPCR can only be used to detect mutations at known sequence positions. ddPCR incorporates two reporter probes, one mutant-specific and one wild-type, because of the requirement to account for PCR-amplification variability among droplets. This approach, by design, allows only the detection of previously known mutations. In cancer, tumor suppressor genes such as TP53 harbor mutations that are scattered throughout the gene as opposed to oncogenes that usually carry mutations located in specific hotspots.¹² Although mutation scanning methods based on amplicon fluorescent melting analysis following real-time PCR have been developed,^13–16 monitoring fluorescent melting within individual droplets during ddPCR is not available at this time. Accordingly, a ddPCR approach that could be implemented in a mutation scanning format is desirable.Here, we demonstrate that incorporation of coamplification at lower denaturation temperature PCR (COLD-PCR),^17–21 within the ddPCR workflow in conjunction with two fluorescently labeled probes matching the wild-type amplicon, provides a simple and robust method for mutation scanning of target amplicons. COLD-PCR suppresses wild-type sequences and enables preferential amplification of mutation-containing droplets, for any mutation along the amplicon.^21,22 Detecting changes to the ratio of COLD-PCR–enhanced signals caused by mutations anywhere within the probed region enables mutation scanning with high selectivity. This novel enhanced ratio of signal-based mutation scanning COLD-ddPCR enables a rapid method for the detection of mutations during ddPCR without prior knowledge of the specific DNA variant present in the target sequence. We demonstrate in this paper the application of COLD-ddPCR to the detection of multiple mutations present in TP53 exon 8, as well as for the T790M resistance mutation in EGFR exon 20 in DNA from mutated cell lines and cell-free circulating DNA (cfDNA) from clinical cancer samples.