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Model Article: Cell nucleus

Outline:

1) Lead Section/Introduction

-Already started with one sentence

-Expand to one full paragraph

2) Discovery

3) Formation/Origin

4) Structure/Identification

5) Use in Assays

INTRODUCTION

A micronucleus is essentially a small nucleus. It usually is a sign of genotoxic events and chromosomal instability. Micronuclei are commonly seen in cancerous cells and may indicate genomic damage events that can increase the risk of developmental or degenerative diseases. Micronuclei form during anaphase from lagging acentric chromosome or chromatid fragments caused by incorrectly repaired or unrepaired DNA breaks or by nondisjunction of chromosomes. This faulty segregation of chromosomes results from hypomethylation of repeat sequences present in centromeric or pericentromeric DNA, irregularities in kinetochore proteins or their assembly, dysfunctional spindle apparatus, or flawed anaphase checkpoint genes. Many micronucleus assays have been developed to test for the presence of these structures and determine their frequency in cells exposed to certain chemicals or subjected to stressful conditions.

DISCOVERY

''Micronuclei are also known as Howell-Jolly bodies because they were first identified and described in red blood cells by hematologists William Howell and Justin Jolly. These structures were later found to be associated with deficiencies in vitamins such as folate and B12. The relationship between formation of MN and exposure to environmental factors was first reported in root tip cells exposed to ionizing radiation. MN induction by a chemical was reported 7 years earlier in Ehrlich ascites tumor cells treated with colchicine.''

ORIGIN OF MICRONUCLEI

Micronuclei primarily result from acentric chromosome fragments or lagging whole chromosomes that are not included in the daughter nuclei produced by mitosis because they fail to correctly attach to the spindle during the segregation of chromosomes in anaphase. These full chromosomes or chromatid fragments are eventually enclosed by a nuclear membranes and are structurally similar to conventional nuclei, albeit smaller in size. This small nucleus is referred to as a micronucleus. The formation of micronuclei can only be observed in cells undergoing nuclear division and can be clearly seen using cytochalasin-B to block cytokinesis to produce a binucleated cells.

Acentric chromosome fragments may arise in a variety of ways. One way is that disrepair of DNA double-strand breaks can lead to symmetrical or asymmetrical chromatid and chromosome exchanges as well as chromatid and chromosome fragments. If DNA damage exceeds the repair capacity of the cell, unrepaired double-stranded DNA breaks may also result in acentric chromosome fragments. Another way eccentric chromosome fragments may arise is when defects in genes related to homologous recombinational repair (ex: ATM, BRCA1, BRCA2, and RAD51) result in a dysfunctional error-free homologous recombinational DNA repair pathway and causes the cell to resort to the error-prone non-homologous end-joining (NHEJ) repair pathway, increasing the likelihood of incorrect repair of DNA breaks, formation of dicentric chromosomes, and acentric chromosome fragments. If enzymes in the NHEJ repair pathway are defective as well, DNA breaks may not be repaired at all. Additionally, simultaneous excision repair of damaged or inappropriate bases incorporated in DNA that are in proximity and on opposite complementary DNA strands may lead to DNA double-stranded breaks and MN formation, especially if the gap-filling step of the repair pathway is not completed.

Micronuclei can also form from fragmented chromosomes when nucleoplasmic bridges (NPB) are formed, stretched, and broken during telophase.

Micronuclei formation may also result from chromosome malsegregation during anaphase. Hypomethylation of cytosine in centromeric and pericentromeric areas and higher-order repeats of satellite DNA in centromeric DNA can result in such chromosomal loss events. Classical satellite DNA is normally heavily methylated at cytosine residues but may become almost fully unmethylated due to ICF syndrome (Immunodeficiency, centromere instability, and facial anomalies syndrome) or after treatment by DNA methyl transferase inhibitors. Since assembly of kinetochore proteins at centromeres is affected by the methylation of cytosine and histone proteins, a reduction in heterochromatin integrity as a result of hypomethylation can interfere with microtubule attachment to chromosomes and with the sensing of tension from correct microtubule-kinetochore connections. Other possible causes of chromosome loss that could lead to micronuclei formation are defects in kinetochore and microtubule interactions, defects in mitotic spindle assembly, mitosis check point defects, abnormal centrosome amplification, and telomeric end fusions that result in dicentric chromosomes that detach from the spindle during anaphase. Micronuclei originating from chromosome loss events and acentric chromosome fragments can be distinguished using pancentromeric DNA probes.

MICRONUCLEUS ASSAYS

The micronucleus test provides important information about a chemical's ability to interfere with chromosome structure and function and many known human carcinogens test positive in mammalian micronucleus tests. In this test, organisms are treated with a chemical and the resulting frequency of micronucleated calls is measured. If there is a marked increase in the number of cells with micronuclei, it can be concluded that the chemical induces structural and/or numerical chromosomal damage. Since micronucleus tests must be performed on actively dividing cells, bone marrow stem cells and the erythrocytes they produce through cell divisions are ideal candidates. These cells experience constant, rapid turnover and the lack of a true nucleus in erythrocytes makes micronuclei easily visible under a microscope.

Micronucleus assay systems are very economical, require much less skill in scoring that conventional metaphase chromosome spreads, and are much faster than conventional tests. Since micronucleus assays reflect chromosomal aberrations reliably and rapidly, they are extremely useful for a quick assessment of chromosomal damage. In particular, the CBMNcyt (cytokinesis- block micronucleus cytome) assay is extremely versatile and is one of the preferred methods to measure the level of chromosomal damage and chromosomal instability in cells. This cytokinesis-block micronucleus (CBMN) assay was first developed to score MN in cells that completed nuclear division by blocking them at the binucleate stage prior to cytokinesis. It later evolved into the CBMN 'cytome' assay to explore more cell death, cytostasis, and biomarkers of DNA damage. The majordrawback in using the micronucleus test is that it cannot determine different types of chromosomal aberrations and can be influenced by the mitotic rate and proportion of cell death, skewing the results.

IDENTIFYING MICRONUCLEI

The number of micronuclei per cell can be predicted using the following formula:

MN/cell = AF/cell * F

AF is the number of acentric fragments and F = 0.5 - 0.5P, where P equals the probability of fragments being included in the traditional nucleus and not forming a micronucleus.

One paper, which used Giemsa stain to stain nuclear material, established the following criteria for identifying micronuclei:

1) diameter less than 1/3 of the primary nucleus,

2) non-retractility (excludes small stain particles),

3) color the same as or lighter than the main nucleus (excludes large stain particles),

4) location within 3 or 4 nuclear diameters or the main nucleus without touching it, and

5) no more than two associated with one primary nucleus (3 or more micronuclei are likely polymorphs or prorubicytes with nuclear fragments).

PATTERNS IN MICRONUCLEUS FORMATION

Multiple studies have found that MN frequency in women is higher than in men and that the number of micronuclei increase until around 70 years of age. Micronuclei levels ranged from 0.5 to 1.4% in men to 0.9 to 1.8% in women. Gender-related differences were mainly seen in younger age groups (<= 50 years) with an almost two-fold difference between men and women. The patterns in the number of micronuclei after 70 years of age is controversial. Some studies have shown that in individuals over 70 years of age, micronucleus frequency increases in both sexes. On the other hand, other studies have found that in the oldest age groups, MN frequencies level off. The deficiency of micronuclei in some of the oldest age groups may be explained by the fact that micro nucleated cells are preferentially eliminated by apoptosis. However, higher MN frequency corresponds to a decreased efficiency of DNA repair and increased genomic instability, which are typical in older subjects. Age-related increases in MN frequency also correspond well with age-related increases in the hypoploidy and the age-related increase in sex chromosome loss. Alternatively, the leveling off of frequency of MN in older subjects would suggest a threshold of genomic instability that cannot be crossed if the person is to survive. If this were the case, women appear to reach this threshold faster than men.

Sex chromosomes contribute to the majority of chromosome loss events with increasing age. In females, the X chromosome can account for up to 72% of the observed MN of which 37% appear to be lacking a functional kinetochore assembly possibly due to X chromosome inactivation. Multiple studies have shown that the frequencies of autosome-positive MN in both genders and of sex chromosome-positive MN in men were similar and remained unchanged in older groups while the frequency of X-positive MN in women was higher than the average frequency of autosome-positive MN and continued to increase until the oldest age.

The frequencies of chromosomal aberrations, damaged cells, and micronuclei are significantly higher in smokers than non-smokers.

Image: http://www.gentronix.co.uk/wp-content/uploads/2012/12/MNT-Diag2.png

Sources:

http://ntp.niehs.nih.gov/testing/types/genetic/invivo/mn/index.html

-National Toxicology Program - US Department of Health and Human Services

http://www.sciencedirect.com/science/article/pii/0165799288900085

-MN/cell = AF/cell * F, where F = 0.5 - 0.5P and P = the probability of fragments being included in the traditional nucleus and not forming a micronucleus.

===AF = acentric fragments

http://ac.els-cdn.com/0027510776901056/1-s2.0-0027510776901056-main.pdf?_tid=7bf44748-8722-11e6-af74-00000aab0f01&acdnat=1475249466_d2087b55f763d2b9539b8b89ba5ddf5a

-Micronuclei are formed when chromosomal fragments are not incorporated into daughter nuclei during mitosis because these fragments lack centromeres.

-Micronucleus assay reflects chromosomal abberation frequencies reliably and rapidly, making it a good for a quick assessment of chromosomal damage.

-One paper, which used Giemsa stain to stain nuclear material, established the following criteria for identifying micronuclei: 1) diameter less than 1/3 of the primary nucleus, 2) non-retractility (excludes small stain particles), 3) color the same as or lighter than the main nucleus (excludes large stain particles), 4) location within 3 or 4 nuclear diameters or the main nucleus without touching it, and 5) no more than two associated with one primary nucleus (3 or more micronuclei are likely polymorphs or prorubicytes with nuclear fragments).

-However, the micronucleus test cannot determine different types of chromosomal aberrations and can be influenced by the mitotic rate and the proportion of cell death.

-Micronucleus assay systems are very economical, require much less skill in scoring that conventional metaphase chromosome spreads, and are much faster than these conventional tests.

http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.584.2198&rep=rep1&type=pdf

-The level of micronuclei is higher in when and in both genders increase with age until around 70 years. MN-levels ranged from 0.5 to 1.4% in men to 0.9 to 1.8% in women. Gender-related difference were mainly seen in younger age groups (<= 50 years) with an almost two-fold difference between men and women.

''-Frequencies of autosome- positive MN in both genders and of sex chromosome-positive MN in men were comparable and remained unchanged in older groups. The frequency of X-positive MN in women was higher than the average frequency of autosome-positive MN and continued to increase until the oldest age.''

-Multiple studies have found that MN frequency in women is higher than in men.

- However, some studies have shown that in individuals > 70 years of age, MN frequency increases in both sexes. On the other hand, some studies have found that in the oldest age groups, MN frequencies level off.

-The deficiency of MN in some of the oldest age groups may be explained by the fast that micro nucleated cells are preferentially eliminated by apoptosis.

-Higher MN frequency corresponds to a decreased efficiency of DNA repair and increased genomic instability.

-Age-related increases in MN frequency correspond well with age-related increases in the hypoploidy and the age-related increase in sex chromosome loss

-Multiple studies have shown that the frequency of X-positive MN in women is higher than the frequency of autosome-positive MN

-The leveling off of frequency of MN in older subjects suggests a threshold of genomic instability that cannot be crossed if the person is to survive. Women appear to reach this threshold faster than men.

http://ac.els-cdn.com/092187349390015U/1-s2.0-092187349390015U-main.pdf?_tid=a319650c-8726-11e6-8324-00000aacb361&acdnat=1475251249_ab3a24a4982ca628793fc4e149ce59cb

-The frequencies of chromosomal aberrations, damaged cells, and micronuclei are significantly higher in smokers than non-smokers.

http://mutage.oxfordjournals.org/content/26/1/125.long

- Scientific Paper - Journal: Mutagenesis (2011)

-micronuclei (MN) are biological signs of genotoxic events and chromosomal instability

- cytokinesis-block micronucleus cytome (CBMNcyt) assay

-two decades - molecular probes and genetically modified cells

-commonly seen in cancer

-indicate genome damage events that may increase the risk of developmental and degenerative diseases

-form during anaphase from lagging acentric chromosome or chromatid fragments caused by incorrectly repaired or unrepaired DNA breaks

''-Micronuclei may result from nondisjunction of chromosomes during anaphase. This faulty segregation of chromosomes results from hypomethylation of repeat sequences present in centromeric or pericentromeric DNA, irregularities in kinetochore proteins or their assembly, dysfunctional spindle apparatus, or flawed anaphase checkpoint genes.''

-MN = Howell-Jolly bodies - identified and described in red blood cells by hematologists William Howell and Justin Jolly - later found to be associated with deficiencies in vitamins such as folate and B12.

-The relationship between formation of MN and exposure to environmental factors was first reported in root tip cells exposed to ionizing radiation.

- MN induction by a chemical was reported 7 years earlier in Ehrlich ascites tumor cells treated with colchicine.

-Cytokinesis-block micronucleus (CBMN) assay was developed to score MN in cells that completed nuclear division by blocking them at the binucleate stage prior to cytokinesis

-developed into the CBMN 'cytome' assay to explore more cell death, cytostasis, and biomarkers of DNA damage.

ORIGIN OF MN

- MN primarily results from acentric chromosome fragments or lagging whole chromosomes that are not included in the daughter nuclei produced by mitosis because they fail to correctly attach to the spindle during the segregation of chromosomes in anaphase. The formation of MN can only be observed in cells undergoing nuclear division and can be clearly seen using cytochalasin-B to block cytokinesis to produce a binucleated cells.

-These full chromosomes or chromatid fragments are eventually enclosed by a nuclear membranes and are structurally similar to conventional nuclei, albeit smaller in size.

- Acentric chromosome fragments may arise in a variety of ways.

-Disrepair of DNA double-strand breaks can lead to symmetrical or asymmetrical chromatid and chromosome exchanges as well as chromatid and chromosome fragments.

- If DNA damage exceeds the repair capacity of the cell, unrepaired double-stranded DNA breaks may also result in acentric chromosome fragments.

-Defects in genes related to homologous recombinational repair genes (ex: ATM, BRCA1, BRCA2, and RAD51) result in a dysfunctional error-free homologous recombinational DNA repair pathway and causes the cell to resort to the error-prone non-homologous end-joining (NHEJ) repair pathway, increasing the likelihood of incorrect repair of DNA breaks, formation of dicentric chromosomes, and acentric chromosome fragments. If enzymes in the NHEJ repair pathway are defective as well, DNA breaks may not be repaired at all.

-Additionally, simultaneous excision repair of damaged or inappropriate bases incorporated in DNA that are in proximity and on opposite complementary DNA strands may lead to DNA double-stranded breaks and MN formation, especially if the gap-filling step of the repair pathway is not completed.

-MN can also form from fragmented chromosomes when nucleoplasmic bridges (NPB) are formed, stretched, and broken during telophase.

-In lymphocytes, MN increase with age and are usually more frequently observed in females than males

-Sex chromosomes contribute to the majority of chromosome loss events with increasing age.

-In females, the X chromosome can account for up to 72% of the observed MN of which 37% appear to be lacking a functional kinetochore assembly possibly due to X chromosome inactivation.

-MN formation may also result from chromosome malsegregation during anaphase.

Hypomethylation of cytosine in centromeric and pericentromeric areas and higher-order repeats of satellite DNA in centromeric DNA can result in such chromosomal loss events. Classical satellite DNA is normally heavily methylated at cytosine residues but may become almost fully unmethylated due to ICF syndrome (Immunodeficiency, centromere instability, and facial anomalies syndrome) or after treatment by DNA methyl transferase inhibitors. Since assembly of kinetochore proteins at centromeres is affected by the methylation of cytosine and histone proteins, a reduction in heterochromatin integrity as a result of hypomethylation can interfere with microtubule attachment to chromosomes and with the sensing of tension from correct microtubule-kinetochore connections.

Other possible causes of chromosome loss that could lead to MN formation are defects in kinetochore and microtubule interactions, defects in mitotic spindle assembly, mitosis check point defects, abnormal centrosome amplification, and telomeric end fusions that result in dicentric chromosomes that detach from the spindle during anaphase.

MN originating from chromosome loss events and acentric chromosome fragments can be distinguished using pancentromeric DNA probes.

ASSAY

-The versatility of the CBMNcyt assay makes it one of the preferred methods to detect the measure chromosomal DNA damage and chromosomal instability phenotype in mammalian and humans cells.

-When cell division is disrupted or chromosomes are broken or damaged by radiations or chemicals, parts of chromosomes may not be included in the daughter nuclei. This DNA not included in the traditional nucleus may form a micronucleus. Micronuclei may also form are when chromosomal fragments are not incorporated into daughter nuclei during mitosis because the fragments lack centromeres.

Bibliography

1) http://ntp.niehs.nih.gov/testing/types/genetic/invivo/mn/index.html

-National Toxicology Program - US Department of Health and Human Services

2) http://www.sciencedirect.com/science/article/pii/0165799288900085

- "A Comment on the Quantitative Relationship between Micronuclei and Chromosomal Aberrations" by John R.K. Savage

-Published in Mutation Research Letters in 1987

3) http://ac.els-cdn.com/0027510776901056/1-s2.0-0027510776901056-main.pdf?_tid=7bf44748-8722-11e6-af74-00000aab0f01&acdnat=1475249466_d2087b55f763d2b9539b8b89ba5ddf5a

-"The Production of Micronuclei from Chromosome Aberrations in Irradiated Cultues of Human Lymphocytes" by Paul I. Countryman and John A. Heddle

-Published in Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis in 1976

4)http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.584.2198&rep=rep1&type=pdf

-"Effects of age and gender on micronucleus and chromosome nondisjunction frequencies in centenarians and younger subjects" by Alina Wojda, Ewa Zie ̨tkiewicz, and Michał Witt

- Published in Mutagenesis in 2007

5) http://mutage.oxfordjournals.org/content/26/1/125.long

- "Molecular Mechanisms of Micronucleus, Nucleoplasmic Bridge and Nuclear Bud Formation in Mammalian and Human Cells" by M. Fenech*, M. Kirsch-Volders, A. T. Natarajan, J. Surralles, J. W. Crott, J. Parry, H. Norppa, D. A. Eastmond, J. D. Tucker, and P. Thomas

- Published in Mutagenesis in 2011

6) http://ac.els-cdn.com/092187349390015U/1-s2.0-092187349390015U-main.pdf?_tid=a319650c-8726-11e6-8324-00000aacb361&acdnat=1475251249_ab3a24a4982ca628793fc4e149ce59cb

- "Cell Division, Chromosomal Damage and Micronucleus Formation in Peripheral Lymphocytes of Healthy Donors: Related to Donor's Age" by Bani Bandana Ganguly

- Published in Mutation Research/DNAging in 1993