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Basic Cytogenetics and the Role of Genetics in Cancer Development
Introduction
This chapter will summarize the knowledge acquired on conventional cancer cytogenetics in the second half of the last century and introduces additional applications of fluorescent in situ hybridization (FISH) available for the study of cancer development and evolution.
Other indications of these techniques applied on cytology samples are also described in Chapter 37 .
Historical Background
As suspected by von Hansemann more than a century ago, cancers are associated with nuclear and mitotic anomalies in their cells.
In 1914, Boveri hypothesized his theory on somatic mutations responsible for the origin and development of malignant transformation. He stressed the acquisition of an unbalanced chromosome constitution as a cause of cancer illustrated by mitotic asymmetry and asynchrony, and foresaw the monoclonal origin of the cancer cell. It took at least 40 more years to establish the exact number of human chromosomes. The blood-culturing method became more successful than the squash method when colcemid was discovered to arrest the mitotic cycle in metaphase by poisoning the mitotic spindle and to prevent the centromeres from dividing. The erroneous adjunction of a hypotonic solution to a pellet of harvested cells was an unexpected improvement in the spread of individualized chromosomes rid of their cellular envelope, resulting in a better dispersal on the metaphase spread.
In 1956, Tjio and Levan accurately reported that the human somatic cell contains 46 chromosomes, including 22 pairs of autosomes and one pair of sex chromosomes; one X of maternal origin and the other chromosome – X or Y – being from the paternal source. Rarely have discoveries had such impact on modern biology and medicine as the description of the 46-chromosome karyotype. The newborn cytogenetic discipline investigated simultaneously the field of inherited diseases and acquired chromosomal anomalies in cancer cells.
Trisomies of chromosome 21 in mongolism and of other autosomes or numerical variations of sex chromosomes proved their specificity and consequently their diagnostic value in congenital syndromes. In 1960, Nowell and Hungerford reported the first evidence of a chromosome anomaly specifically associated with a malignant disease, the chronic myelogenous leukemia. They showed the recurrent presence in leukemic leukocytes of a deleted small chromosome that they named the Philadelphia (Ph) chromosome in reference to the city where they were working. This proof of a genetic cause in cancer was the starting point to new insights into the pathways of malignant initiation and progression.
Basic Knowledge of Cytogenetics
The human somatic cell contains two copies of each chromosome, one from paternal and the other from maternal origin. Therefore the karyotype is diploid with double the amount of DNA (2 n ) compared with the gametes ( n ) with a single set of 23 chromosomes.
The first step is to review the different stages of the cell cycle, which are essential to the acquisition of chromosomes suitable for karyotyping.
Cell Cycle
The cell cycle is a process of successive cell divisions (mitosis) interrupted by so-called “resting” periods (interphase). Actually, the resting cell is very active metabolically, with continuous molecular interactions between DNA, RNA, and proteins.
The Interphase
The interphase is the period when the cell is in a non-dividing state and this can be in different stages: the first gap (G1) between the last mitosis and the S phase (phase of DNA synthesis) and the second gap (G2) between the completion of the S phase and the next mitosis (M). The mitotic division occupies only a short time in the cell cycle. If the cell reaches its ultimate stage of differentiation and will not divide anymore, the cell is said to be in phase G0 of the cycle. G0 applies also for the cells that have temporarily stopped dividing ( Fig. 2-1 ).
During the G1 phase, the cell is metabolically active and requires many organelles for protein synthesis, while acquiring the potential for the DNA doubling process. The duration of the entire cycle depends on the time of the G1 phase, which varies according to different conditions and tissue types. The G1 phase may last from only a few hours to weeks or months, depending on the mitotic rate of the tissue. The phase of DNA synthesis (chromosome replication) has a duration of approximately 8 hours. The replication is not homogeneous throughout the genome, and asynchronism of replication occurs, particularly in the synthesis of the heterochromatin composing the inactivated X chromosome.
DNA replication is achieved when all the chromosomes are duplicated in two identical sister chromatids with the consequence that the total amount of DNA is now doubled if compared with the normal 2 n value of the interphase nucleus. The following phase, G2, takes about 4 hours and accumulates the cytoplasmic organelles necessary to complete the mitosis.
This step-by-step progression is controlled by a series of checkpoints which stop the process if the previous phase is not achieved. Different proteins act sequentially on the cell cycle; the cyclin-dependent kinases (CDKs), the cyclins, and the CDK inhibitors (CKIs).
Activation of kinases by cyclins positively regulates the cycle by allowing the cell to enter the successive phases. If the quality of DNA synthesis is impaired, CKIs would automatically stop the process and drive the cell to apoptosis.
The Mitosis
Although the cell cycle is a continuous process, four distinct phases are described in mitosis ( Fig. 2-2 ).
Prophase
Condensation and fragmentation of the chromatin into chromosomes start to make them visible. The nucleolus vanishes and the centrioles, replicated in G2, migrate to opposite poles of the cell. Each chromosome is still attached to the nuclear membrane and composed of a double strand of sister chromatids. A constricted area called a “centromere” becomes apparent on the chromosomes and the nuclear membrane disintegrates.
Metaphase
The chromosomes are aligned at the equatorial plate of the mitotic spindle and attached by their centromere to the network of microtubules. Metaphase chromosomes are composed of two sister chromatids joined together by the centromere.
Anaphase
The centromeres are split into two parts and attract each single strand of the sister chromatids to one pole by shortening of the spindle fibers. The chromosomes, pulled apart, are clustered at each pole of the cell.
Telophase
Telophase results in the formation of a nuclear membrane. The constriction of the cellular membrane starts the division of the cytoplasm (cytokinesis). The chromosomes progressively melt back into a chromatin network. At the end, both daughter cells have the same number of chromosomes as the maternal cell.
The Meiosis
Meiosis is a more complex process by which the gonad cell undergoes two cellular divisions.
The meiosis I follows stages similar to the mitotic division. During the prophase I, each chromosome is duplicated. Chromatid exchanges occur between paired homologue chromosomes, which are linked together by their sites of junction: the chiasmas. This process, called “crossing over,” results in genetic recombination, with the consequence that genomes between maternal and daughter cells will not be strictly identical. Anaphase I starts with the migration of homologue chromosomes to the opposite poles of the cell without splitting of their centriole. Meiosis II arises without previous DNA synthesis and produces the longitudinal separation of the two chromatids, therefore reducing the cell to a haploid n number of 23 single-stranded chromosomes. The fecundation of the ovule by the spermatozoid will restitute the diploid value of somatic cells and provide a complete zygotic genome.
The Chromosome Structure
The chromosomes are composed of DNA and associated histone and non-histone proteins.
This combination called “chromatin” is individualized into visible chromosomes only during mitosis. The double helix of DNA, described by Watson and Crick, is supercoiled around protein cores in a complex structure of nucleosomes. Compacted nucleosomes constitute chromatin segments of approximately 30 nm in diameter observable in electron microscopy. Further condensation makes it optically identifiable as heterochromatin in the interphase and as chromosomes at the late prophase. An animation on cell division and chromosome structure can be found at http://www.johnkyrk.com .
The extremities of the chromosomes are called telomeres. They preserve the integrity of chromosomal extremities by allowing replication to occur without loss of coding sequences, but undergo repetitive shortenings themselves after each cellular division. The so-called “mitotic clock” counts the number of cell divisions that have occurred and pushes the cell to apoptosis before a critical telomeric shortening is reached. If this should occur, chromosomes would be prone to fuse end-to-end, giving rise to sticky ends that would favor mitotic aberrations and promote the accumulation of subsequent genetic rearrangements, possibly leading cells toward the first crucial steps in the development and progression of neoplasia.
Methodology
The Karyotype
The 22 pairs of autosomes have been classified into seven groups: A–G, according to their length and the position of the centromeric constriction. The largest pairs are numbered 1–3 in group A. The centromere is located in the middle of chromosomes 1 and 3 and displaced in a submetacentric position in pair 2. Group B is composed of pairs 4 and 5, both with a subtelomeric centromere. Group C is the largest one and is composed of medium-sized chromosomes including pairs 6–12 and chromosome X. Most of them are submetacentric and roughly classified by decreasing length. Group D is composed of chromosome pairs 13, 14, and 15 and characterized by a distal acrocentric centromere. Group E contains the metacentric pair 16 and the submetacentric 17 and 18 sets. Chromosome pairs 19 and 20 are smaller metacentric chromosomes and constitute the group F. Group G is composed of small acrocentric chromosomes arbitrarily placed in pairs 21 and 22. The small Y chromosome is included in this G group.
Accurate individual classification of chromosomes was rendered possible by the banding techniques first developed by applying fluorescent quinacrine mustard on metaphase preparations. This fluorescent agent reveals transverse bright bands (Q-banding) of different intensities along the chromosome arms. Other procedures using trypsin digestion and Giemsa staining yield dark G-bands superimposable on the bright Q-bands. This led to a very precise identification of each individual chromosome ( Fig. 2-3 ). Techniques with heat denaturation in saline solution obtained a reverse staining called R-bands with optional enhancing of telomeric ends in T-banding.
The different banding pattern for each of the 23 different chromosomes allows perfect pairing of homologues. The number of bands can be raised to 800 by the high-resolution staining technique, obtained on prometaphase chromosomes. The dark G-bands correspond to a compact conformation of the chromatin, while the clear bands are composed of uncoiled chromatin. The dense Q- and G-bands contain repetitive inactive DNA. Active genes are supposed to be in clear bands; constitutive heterochromatin is located in the pericentromeric regions as revealed by C-banding and appear as chromocenters in the non-dividing nucleus. Chromosome Y has a unique strong fluorescent appearance visible in the interphase nucleus as a bright dot, also visible as a dark C-band. With these staining methods, the chromosomes 21, already recognized in the pre-banding era because of their known involvement in Down syndrome, remained classified as such, and the minute marker of CML was consequently considered as belonging to pair 22.
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