DNA Repair Genes

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					Chapter one                           Introduction and Literature Review

1.2. Literature Review
1.2.1. Cancer
   Cancer is defined as an abnormal mass of tissue, the growth of which
exceeds and is uncoordinated with that of the normal tissues, and persists
in the same excessive manner after cessation of the stimuli evoked the
change (Kufe et al., 2003). Cancer cells can divide continuously if given
sufficient nutrients and space; this is vividly illustrated by the cervical
cancer cells of woman named Henrietta lacks, which died in 1951
(Rastogi, 2003). Tumors are classified according to their behavior and
histogenesis (cell of origin) in to benign and malignant types (Cortran et
al., 1999). Benign tumors are localized and consist of well differentiated
cells similar to the tissue of origin; such tumors never establish growths
in the other parts of body; however, these tumors may sometimes become
harmful and lethal if they are located in organs such as brain and liver
(Underwood, 2004). Malignant tumors are in fact cancerous growth,
usually invasive type of tumors, derived from single cell, thus
monoclonal is character (Noruzinia et al., 2005). The aberrant cell is
usually embryonic type, undifferentiated, having large irregular nucleus
and deficient in cytoplasm compared with the normal cells; such cells
proliferate uncontrollably and have an infinite growth and doubling
potential until death (Cortran et al., 1999). Another important aspect of
malignant tumors is their property of Metastasis (Lewis, 2004).
Malignant cells are detached from the malignant neoplastic growths and
get distributed to other locations in the body through circulation where
they establish secondary tumors; Metastasis is a major feature of
malignant growths, which makes their surgical removal almost
impossible (Alizadeh et al., 2001).

Chapter one                            Introduction and Literature Review

1.2.2. Carcinogenesis
    Carcinogenesis is a multisteps, multigenic and multicausal process
(Nguyen and Vasseur, 1999). Mutational mechanisms can be proposed
for most; if not all, known human carcinogen (Toft and Areneds, 1997).
    Many of these are electrophilic or metabolically activated to reactive
molecules which can alter DNA, causing genetic damage and different
types of mutation, even some human carcinogens previously proposed to
be non-genotoxic (e.g. Hormones and asbestos) exhibit mutational
activity in assays for chromosomal mutation (Wei et al., 2000).
Carcinogenesis has three stages which are the following:- It is the first step in the formation of a tumor that it
begin at the cellular level when enough mutation accumulate in genes
controlling proliferation and other processes within a single clone of
cells, then the cell loss their essential fracture (Squartini, 1996). Promotion: In this stage mutant cell converted to cancer cell in
processes called transformation by external fact of named promoted, that
make the cell loss their differentiation (Parker and Kim, 2005). Progression: At this stage the primary tumor would be formed,
and this tumor do not developed with out supply with blood (Pitot and
Dragan, 1991). If this primary tumor do not removed, tumor releases
factors that bind to endothelial cells of nearby capillaries and stimulate
them to invade surrounding area (extra cellular matrix) and proliferate
(divide) toward tumor (Bardi et al., 1997). New blood vessel extension
nourishes tumor; all form of cancer cell spread with the help of collagen
dissolving mechanism, to reproduce and spread to other parts in the body
(Hunnings et al., 1993).

Chapter one                           Introduction and Literature Review

1.2.3. Causes of Cancer Chemical factors
   Many chemical carcinogens have now been identified; the
carcinogenic risk cannot be predicted from the structural formula alone,
even apparently closely related compounds can have different effects
(Kufe et al., 2003). Some agents act directly, requiring no metabolic
conversion, others (pro-carcinogens) require metabolic conversion
(ultimate carcinogens) (MacSween and Whaley, 1997).
    In principle, chemical carcinogens of these types are frequently
capable of eliciting pleiotropic cellular changes at the genetics and
epigenetic levels, and can divided in to two groups:-
1) DNA-damaging agents that induce formation of DNA adducts and to
 subsequent mutation, known as somatic mutation (Peto et al., 1975;
 Fearon and Vogelstein, 1994).
2) Those that alter cellular signal translational modification (Barrett,
   Changes in the first group (i.e. somatic mutation) are permanent,
whereas those in the second group (Post-translation) changes are
transient; the effects of environmental factors at the protein level also are
transient as a result of the limited life-span of a given protein, both types
can alter control of cell growth or death (Yuspa et al., 1994). Physical factors
    There are many examples of physical factor, like rays: there are
many sources for rays that lead to the formation of cancerous and this
depended on the exposed period and type of ray like: Ionizing radiation
that when human exposed it lead to cancer of skin (Woolf, 2001).

Chapter one                          Introduction and Literature Review Biological factors Bacterial infections
    Biological factors; accumulate epidemiological and clinical data have
identified Helicobacter pylori as a risk factor for gastric carcinogenesis,
infection with H.pylori is more prevalent in Asian population than in
Western population, particularly in Japan and Korea where gastric cancer
is the most frequently occurring malignancy (Marchetti et al., 1995). Viral infections
    Some forms of cancer are caused by tumor viruses (Russell, 1998).
Insertion of viral genome in to host cell genome may either cause
abnormal promotion of cellular proto-oncogen leading to neoplasia, or
transcription and expression of viral oncogene leading to neoplasia
(Steven and Lowe, 2000). Chronic hepatitis B virus infection is
epidemiologically    associated    with    hepatic   cellular   carcinoma
(Underwood, 2004). Fungal infections
   Maki and colleagues (2001) reported that Mycotoxins are toxic
substances produced by fungi, those having the greatest relevance in
human carcinogenesis are aflatoxins produced by Aspergillus flavus.
Aflatoxins, particularly aflatoxin B1, are among the most potent
carcinogens and have been specifically linked to high incidence of
hepatocellular carcinoma in certain parts of Africa (Denissenko et al.,
2000). Parasitic infections
    Parasitic infections are highly prevalent in the general population,
detecting a relationship between a parasitic infection and cancer is not an
easy task; it requires epidemiological, microbiology and molecular

Chapter one                             Introduction and Literature Review

biology techniques (Abdel-Rahim, 2001). Few parasites, e.g., Shistosoma
haematobium and Opisthorchis viverrini have been found to be strongly
associated with bladder cancer and cholangio carcinoma respectively
(Khurana et al., 2005). Hormonal factors
    Estrogens can be shown experimentally to promote the formation of
mammary and endometrial carcinoma, the association between breasts
carcinoma and oral contraceptive containing estrogens remains unproven
(Underwood, 2004).
   Ripple and his colleagues (1999) hypothesized that androgen
exposure, which has long been associated with the development of
prostate cancer, may be means by which the pro-oxidant antioxidant
balance of prostate cells is altered. Genetic factors Oncogenes: Genes that normally trigger cell division are called
proto-oncogenes; they are active where and when high rates of cell
division are necessary, such as in a wound or in a rapidly growing
embryo (Ramzi et al., 1999). Sandberg (1999) reported that when proto-
oncogenes are turned on at the wrong time or place, they function as
oncogenes and cause cancer ("onco" means cancer). This abnormal
activation may be the result of a mutation, a single base change in a
proto-oncogene causes bladder cancer, for example (Eisenman and
Grandori, 1997). Any changes that had been happen in the proto-
oncogenes results from the carcinogenesis factor leads to change it in to
oncogenes that activated the formation of tumor by increases the gene
expression (Ramzi et al., 1999), that happen through the following
points :-

Chapter one                          Introduction and Literature Review

A. point mutation: The oncogene thought to be responsible for human
bladder cancer differs from its proto-oncogene counterpart by a single
point mutation that results in a glycine codon to be changed to a valine
codon (KolasekandPeter, 1997)                                            .
B. gene amplification: In this situation a gene is copied over and over,
often as sequential repeats; this repeated copying produce more and more
gene product; like in myc gene when the oral keratinocytes infection with
papilloma virus (Lieberman and Lebovitz, 2001)                           .
C. chromosomal aberration: Like translocation happen in the myc gene
from chromosome number 8 to chromosome number 14 that leads to
Burkitt lymphoma in children (Sandberg, 1999)                            .
D. gene fusion: like in Philadelphia chromosome results from fusion in
chromosome 9 and 22 that causes chronic myelogenous leukemia (CML)
(Micheal and Leland, 2000). Tumor Suppressor Genes: - Some cancer result from loss of a
gene that normally suppresses tumor formation; the normal state tumor
suppression results when cells respond to growth inhibiting signals
(Shovlin et al., 1999). Where as oncogene activation is usually associated
with a point mutation, chromosomal translocation or inversion, and a gain
of function, a tumor suppressor gene mutation that causes cancer is
typically a deletion, which removes a function (Stean, 2000). Certain
DNA viruses, such as SV40, adenovirus, and human papilloma virus, are
also associated with a high risk of developing particular cancers; these
viruses interact with the normal products of tumor suppressor genes
(Martin, 1999). Among the best-studied tumor suppressor genes are the
retinoblastoma gene, the p53 gene, and BRCA1 (Gorgoulis et al., 2000).

Chapter one                          Introduction and Literature Review

    If a cell loses a P53 gene, or if the gene mutates and no longer
functions properly, the cell may lose cell cycle control and become
cancerous; mutational analysis and epidemiological observations reveal
that P53 may be a genetic mediator between environmental insults and
development of cancer (Anthony et al., 2000). The BRCA 1 gene, which
stands for "breast cancer predisposition gene 1" greatly, increases the
lifetime risk of inheriting certain forms of breast and ovarian cancer;
BRCA 1 was linked to a marker on chromosome 17 in 1990, and then
mapped and sequenced in 1994. It is a tumor suppressor gene, because
the phenotype results from a loss of function (Lewis, 2004). DNA Repair Genes:- These genes found normally in the cells
and have an important role in the growth, division and differentiation
processes; deficiencies in DNA damage signaling and repair pathway are
fundamental to the etiology of most human cancer (Kufe et al., 2003).
   Kolasek and Peter (1997) found that, DNA exposed to variety of
different damaging agents, which can be of either exogenous or
endogenous origin. No single repair process could manage with the great
variety of genetic lesions, and multiple, partly overlapping damage repair
pathways are therefore required in mammals (Hoeijmarker, 2001). The
major mechanisms of DNA repair include excision, recombination and
mismatch repair (Bronner, 1998).
1.2.4. Treatment of cancer Surgical therapy
   Surgery is the oldest treatment for cancer (Barclay, 2000). Complete
surgical excision represents the most effective therapy for most solid
tumors (Rosenberg, 1999). Yet, failure to remove regional lymph nodes,

Chapter one                          Introduction and Literature Review

local recurrence, distant metastasis, beside significant cosmetic or
functional defect, are the main limitations of surgery (Siguardeson et al.,
2001). Radio therapy
    Treatment of tumors by X-rays is an important line of therapy. Since
cancer cells are rapidly dividing, radiation causes chromosomal breakage
and destruction of cells, although some normal cells are also destroyed
during the treatment. Radiation may only cause a temporary remission of
cancer. Excessive exposure to radiation can cause systemic imbalances
that could lead to death (Rastogi, 2003). Chemo therapy
   The introduction of chemotherapy in the fifties and sixties decades of
the last century has resulted in the development of the curative
therapeutic interventions for patients with several types of solid tumors
and hematopoietic neoplasm (De Vita, 1997). Types of chemotherapy
generally used as reported by (Rugo, 1994) are:-
I. Induction chemotherapy in which drug therapy given as the primary
  treatment for the patients.
II. Adjuvant chemotherapy where drug therapy is given after the control
  of tumor by an alternative method.
III. By site-directed perfusion of specific regions of the body most
   affected by cancer. Bio therapy
   Biotherapy is the use of agents derived from biologic source of
approaches that affect the body biologic responses (Aberanthy, 2000).
They act through modifying the relationship between the tumor and the
host leading to modulation of the host's biologic response against the

Chapter one                             Introduction and Literature Review

tumor cells, and thus achieving therapeutic benefit (Mihich and Fefer,
1983). Immune therapy
       Immunotherapy involves producing anti-tumor effects primarily
through the action of natural defence mechanisms; it is believed that the
immune system plays a major role in rejecting tumor cells, which are
foreign relative to normal cells (Gaynor and Fisher, 2000). Biological
response modifiers (BRMs) work on boosting the body's immune system,
The majority of BRMs are classified as cytokines; BRMs are used not
only to inhibit tumor growth, but also to alter the interaction between the
cancer and the body's defense mechanism (Lewis, 2004). These include:
   - Hematopoietic growth factors (HGFs) – includes colony
         stimulating factors (CSF), which encourage the growth of bone
         marrow stem cells (Hillman, 2005).
   - Interleukin (ILs) - these naturally occurring lymphokines can
         stimulate activity and growth of the body's immune cells, such as
         lymphocytes (Pardoll, 1993).
   - Interferon (IFNs) - these naturally occurring cytokines boost the
         body's immune response and act directly on cancer cells to control
         their rapid growth (Ahn et al., 2003).
   -     Monoclonal antibodies (MAB s) - antibodies that are produced in
         a laboratory setting by injecting cancer cells in to mice and
   - Extracting the cells that make antibodies to fight off the cancer
         (Frank et al., 2005). Gene therapy
       It is defined as the modulation or transfer of genetic material with
therapeutic objective (Lewis, 2004). Malignant cells harbor genetic

Chapter one                             Introduction and Literature Review

mutations that are believed to be responsible for neoplastic, phenotype
this may be amenable to correction through gene transfer approaches
(Cornetta and Robertson, 2000). The transfer of target gene in to a cell
could be directed through transfection, or indirect through transduction
via virus vector system (Kufe et al., 2003). Approaches of gene therapy
in cancer treatment are (Aberanthy, 2000):
   - Gene replacement - supply cell with healthy copies of missing or
        flawed genes.
   -     Enhanced cytokine therapy - deliver cytokines by inserting their
        gene in to either tumor cell or cells that infiltrate tumors.
   -     Drug targeting therapy - inject a tumor with a gene that renders the
        tumor cell vulnerable to a drug.
1.2.5. Complementary and Alternative Medicine
       The term complementary and alternative medicine refers to products
and regimens that individual may employ either to enhance health and
well being or cure diseases, it's current popularity among conventional
medical treatment was in patients with life threatening diseases such as
cancer (Boom et al., 2000). Herbal medicine is a growing area of health
care that demands attention; it is also an important branch of alternative
medicine (Choudhary and Atta-Ur-Rahman, 2004). Plants have a
significant role in maintaining human health and improving the quality of
human life for thousands of years, it was documented that herbs were
used for medicinal purposes for centuries (Craig, 1999). There are at least
250,000species of plants have been found to possess significant
anticancer properties (Ashis et al., 2006).
   Three major types of chemoprevention agents of plant origin have
been identified, namely inhibitors of carcinogen formation, blocking

Chapter one                           Introduction and Literature Review

agents and suppressing agents or anti-progression agents (Nandi et al.,
   Scientists have been shown that the uses of organic solvent like
hexane, methanol and acetone, in the plant extraction less the mutation
level, also have been shown that the extracted plant compound by the
hexane is anti-mutant activity (Nakamura et al., 2001).
   Some of the best cancer drugs on the market today originally came
from plants, which is used to treat tumors including breast, ovarian, and
non-small cell lung cancer (Lee et al., 2000). One of these active
chemical compounds derived from plant and plays an important role in
cancer therapy is flavonoid which is toxic to cancer cell line or to
immortalized cells, but are less toxic to normal cells (Nijveldt et al.,
2006). The cytotoxic effect of flavonoid isolated from the aerial parts of
the Asteraceae family was studied in vitro using malignant human
rhabdomyosarcoma (RD) cells and on normal African green monkey
kidney (Vero) cells, the results showed that the natural compound have
selectivity (Naz et al., 2006).
   Ellipticine is a pyridoindole alkaloid isolated from the leaves of
several Ochrosia species, the toxic side effects associated with Ellipticine
prompted efforts to improve its pharmacological profile that lead it to
cancer chemotherapy (Kraut et al., 2006). Isolated compounds like
bryonolic acid (from Trichosanthes kililowii), crocin (from saffron) and
allicin (from Allium sativum) have also been found to induce apoptosis
and therefore arrest cell proliferation (Campbell et al., 2002).

Chapter one                           Introduction and Literature Review

1.2.6. Meliaceae
     Meliaceae, or the mahogany family, is a flowering plant family of
mostly tree and shrubs (and a few herbaceous plants) in the order
sapindales, characterized by alternate, usually pinnate leaves without
stipules, and by syncarpous, apparently bisexual ( but actually mostly
cryptically unisexual ) flowers borne in panicles, cymes, spikes, or
clusters (James and Welsh, 2003). Most species are evergreen, but some
are deciduous, either in the dry season or in winter, the family includes
about 20 genera and 550 species, with a pan tropical distribution, one
genus (Toona) extends north in temperate china and south in the
southeast Australia (Singh and Srivastava, 1996).
    Most famous compound that has been discovered which is:-
Azedirachtin, Meliantriol, Limonoid and Salannin . One of the most
important species is Azedarachta and Melia (Balandrin et al., 1991).

1.2.7. Melia azedarach
     This tree has many common names such as:- China berry, China
tree, pride of India, Indian lilac, Persian lilac, bead tree, Taxis umbrella,
This elegant tree is cultivated in the warm climates of Europe and
America also M. azedarach grown in different area in Iraq; it dose not
grow to any extent north of Virginia, flowers early during the spring.
"But the fruit is toxic only if large quantities ate"(Dymock, 2001).

Chapter one                           Introduction and Literature Review

     Figure (1.1) Melia azedarach plant grown in a local garden in
                               Baghdad. Biological and Ecology
     Little has been written on the ecology of M. azedarach based on
general description of habitat, it is likely that M. azedarach requires open
sun; and in part shade, and is adapted to awide range of soil moisture
condition, in South Africa; M. azedarach has spread a long road side
(Henderson and Musil, 1994). Horticultural references indicate that M.
azedarach is fast growing, it can reach 6-8 meters in height within four or
five years, maximum height can be 12 – 16 meters. In comparative
studies of plant growth in India (Dennis, 2007). M. azedarach completed
most growth during the initial dry part of the growing season, indicating
that it uses reserves from the preceding year for growth. M. azedarach
also has a shallow root system, generally within the top 70 cm of the soil,
and allocates most of its photosynthetic in above ground shoots (Bisht
and Toky, 1993).

Chapter one                           Introduction and Literature Review Medical Importance and uses of Melia azedarach
     Many scientists have been study the use of different parts of M.
azedarach for medical purposes (Paula et al., 2002). The plant extract
used as antiviral like in Herpes virus infection, this may be due to the
presence of Meliacine (MA) which is a peptide compound that are
separated from the leaf that have a highly activity against the viruses
Herpes virus infection type -1- (HSV-1-) (Villiamil et al., 1995).
Meliacine (MA) has inhabit the replication of nucleic acid (DNA, RNA)
for many in vitro animal viruses, that the formation of the Interferon
(IFN) which is antiviral substance that formed by the cells which is
infected with viruses reduced when cell treated with meliacine (Andrei et
al., 2000).
    Kim and others (2000) have been extract 28- deacetylsendanin (28 –
DAS) that have antiviral activity against (HSV-1-), the inhibitory
concentration (50 %) of viruses of this compound is (1.46 µg/ml), and at
the concentration (400 µg/ml) does not has any toxicity in vitro. In
another study, they found that the MA compound which is separated from
the leaf of M. azedarach inhabit the replication of foot and mouth disease
virus (FMDV) in the cells (BHK-21) (Wachsman et al., 2000). Also have
been found that MA compound which separated from leaf inhabit the
replication of viruses in vitro in the cells treated with this compound
pretreatment or directly after virus adsorption, so it would be clear that
the MA inhabit virus infection (Castilla et al., 1998).
    MA, an antiviral activity present in crude leaf extract that inhabits
HSV-1- multiplication in vitro, was studied in a murine; herpetic stromal
keratit is experimental model. Adult Balb/c mice were inoculated with
HSV-1- at their corneas after abrasion (Andrei et al., 2000).

Chapter one                             Introduction and Literature Review

    As example for the using M. azedarach as a therapeutic action was
seen in the SITK company which used about 15.9 mg per tables of M.
azedarach, that the effectiveness of it has been carefully monitored and
proven via rigorous and extensive clinical testing; clinical trials also
prove that it can handle all forms of acne, including severs, cystic acne,
acne vulgaris without any side effect
(Hussein and Karim, 2006).
   They have found a different uses of M. azedarach, which they used
the aqueous extract of steam in treatment of asthma, and they used the
boiling extract of root as antipyretic for human (Watt and Brandwijk,
1962). In the south of America they have been used the leaves extract to
in heal eczema, treat muscle pain, leprosy and, also nervous pain
(Sandeep verma and Dandiya, 1990). M. azedarach and another plants
which is belong to the Meliaceae family are very important because it
contain an active compound belong to the Tetranortriterpenoid in
different parts of plant and from these compound which have a specific
importance, Meliatoxins (A1, A2, B1, B2) that is separated from M.
azedarach (Bhuiyan et al., 2001). Orally, M. azedarach leaf is used for
eye disorders, epistaxis, intestinal worms, abdominal upset, anorexia, skin
ulcers, cardiovascular disease, contraception, abortion, diabetes, fever,
gingivitis and hepatic dysfunction (Dandiya, 2001). The bark
(azedarachtin compound) is used for malaria, peptic ulcer, skin diseases,
pain, and fever; M. azedarach seed oil also might have immune stimulant
effects, it seems to stimulate cellular immune response (Lioyd and Felter,
1998). Subapriya and colleagues (2005) have evaluated the modifying
effects of ethanol extract of M. azedarach leaves on oxidative stress
induced by the potent gastric carcinogen N-methyl-N-nitro-N-

Chapter one                           Introduction and Literature Review

nitrosoguanidine (MNNG) in male Wistar rats. The extent of lipid
peroxidation and the status of the antioxidants superoxide dismutase
(SOD), catalase (CAT), reduced glutathione (GST) and glutathione
peroxidase    (GPx)    were    used    as   intermediate   endpoints   of
chemopreventation (Wargovich et al., 2001).
   Schwartz (2006) is currently studying is flavopiridol, which was
originally isolated from the bark of a tree M. azedarach, this compound, a
cell-cycle inhibitor that enhances apoptosis (programmed Cell death), is
now manufactured in the laboratory Flavopirdol also have their activity
against cancer by stopping the cell division in the G1 and G2 phase by
interaction with the cyclic depend kinas then it would be stopped the cell
division (Edenharder et al., 2002).
1.2.8. Groups of Chemical Secondary Metabolites
    The chemicals secondary metabolites are very variant and complex
chemical compound (Abuharfeil et al., 2000). The classifications of these
compounds are different, some scientist classified it according to the
chemical structure of these compounds, and others classified it according
to the source of metabolism process (Hartmann, 1996). And the most
common compounds are:- Alkaloids
    Alkaloids extremely difficult to define because they do not represent
a homogeneous group of compounds from the chemical, biochemical, or
physiologic viewpoint (Verpoorte and Alfermann, 2000). Consequently,
expect for the fact that all organic nitrogenous compounds, plants have
been a rich source of alkaloids, but some are found in animals and fungi
(Oomah, 2003). The essential elements of any alkaloids are hydrogen,
carbon, oxygen, and nitrogen (one atom or more) some of them are in

Chapter one                            Introduction and Literature Review

aqueous state like nicotine (Harborne, 1993). Phenolic compounds
    This group consists of thousands of diverse molecules with
heterogeneous structure, with common feature of having one or more
phenol rings (Harborne, 1973). Most of the phenolic compounds belong
to flavonoids, a group of polyphenolic compounds with 15 carbon atoms
(15 C) based on a skeletal structure of two benzene rings joined by a
linear C3 chain (C6 – C3 – C6 – system) (Taiz and Zeiger, 2002).
   Flavonoids compounds are highly characteristic to plants; many of
flavonoids are easily recognized as the pigments in flowers and fruits, and
occur in all parts of plants (Sawain, 1998). Volatile oil
   Volatile oils are odorous principle found in various plant parts; it is
called volatile oil because it is evaporated when exposed to the air, at
ordinary temperature (Cabrera, 2001). Volatile oils are colorless when
they are fresh but on long standing, they may oxidize and darkened in
color; to prevent this they should be stored in a cool dry place in tightly
closed glass container (Conn, 1981). Terpenoid compounds
    Trease and Evans, (1989) reported that Terpenoid are hydrocarbon
compounds that connect essentially to a number of Isoprene units, the
common rules to this compound are (C5 H8)n. Terpenoid are aromatic
chemical compounds which is dissolved in fates, its found in the
cytoplasm of plant cells or in special glands like the volatile oil, or it
could be found in green plastids or carotenoids (Harborne, 1993). One of
the most important terpenoid is azedarachtin that is separated from
Meliaceae, this compound have a high toxicity against another organism

Chapter one                           Introduction and Literature Review

(Mordue and Black well, 1993). Azedirachtin is found             in the M.
azedarach tree and Azedarachta indica, the major constituent is
azedarachtin A and other isomers that may be present including
azadirachtin are azadirachtins H, B and C; the cytotoxicity of
azadirachtin A in several cultured human glioblastoma cell lines has been
examined (Akudugu et al., 2001). Resins
    Chemical compound with a high complexly in their composition,
formed from the oxidation of different volatile oil, and formed in special
vacuoles in the plant and usually found at the surface of the bark that free
zed when exposed to air (Conn, 1981). It could be found alone or
company with another oils ,another characteristic of the resins that it
could not dissolved in the water but it could be dissolved in either and
any another organic solvent (Harborne, 1993). Saponine
    Saponine are special glycosides that have a foam like even if its
found at a low concentration, these compound contain non sugar (a
glycol) part and this part called sapogenin, most of them are steroid
compound in the plant kingdom and it has been found in different parts of
the plant but, its quantity affected by the level of plant growth (Saad,
1997). Glycol part formed by the acidic or enzymatic analysis, these
compound are highly toxic for the murine, also sapoinin are used as
insecticide (Edward and charles, 1999).

Chapter one                            Introduction and Literature Review

1.2.9. Plant Tissue Culture
   Plant tissue culture some time called Micro propagation is the
technique of growing plant cells, tissues and organs in an artificial
prepared nutrient medium semi solid or liquid, under aseptic conditions;
better control of environmental conditions like light, temperature, gas
mixtures and nutrients can be achieved for plant tissues growing in vitro
(Barnum, 2003). At present, plant tissue culture technique has advanced
to a level where it can be applicable to economic problems; our
knowledge of cell and tissue cultures has been developed with full swing,
especially in biotransformation, genetic engineering, and secondary
metabolite (Zafar and Datta, 1992).
1.2.10. Stages of Micro propagation Source of explants
   Explants quality and subsequent responsiveness in vitro are
significantly influenced by the phytosanitary and physiological condition
of the donor plant (Pierik, 1987). Prior to culture establishment, careful
attention is given to the selection and maintenance of the stock plants
used as the source of explants; stock plants are maintained in clean,
controlled condition that allow active growth but reduce the probability of
disease (Purohit et al., 2003).
    Maintenance of specific pathogen tested stock plants under condition
of relatively lower humidity, and use of drip irrigation and antibiotic
spray have proved effective in reducing the contamination potential
candidate explants (Trigiano, 2000). Sterilization of explants
   The sterilization of explants is the most important part in the success
of plant tissue culture program. The sterilization process meaning to get

Chapter one                           Introduction and Literature Review

ride of microorganisms that their presence lead to kill the explants either
by their speed growing or by their toxic material produce (Omer and
Mohamed, 1998). The time of sterilization and the material have been
used to sterile the explants differ from plant to another, that have been
found that the uses of ethanol (15%) for two minutes and sodium
hypochlorite (2%) to sterile the Brassica compestris leaves give a good
result (Peak et al., 1990). Callus
   The first step in many tissue culture experiments, it is necessary to
induce callus formation from the primary explants; these explants may be
an aseptically germinated seedling or surface sterilized roots, stems,
leaves, or reproductive structures (Sala, 2000). Callus is basically more
or less non-organized tumor tissue which consists of an amorphous mass
of loosely arranged thin-walled parenchyma cells arising from the
proliferating cells of the parent tissue (Esau, 1999). The general growth
characteristics of a callus involve a complex relationship between the
plant materials used to initiate the callus, the composition of the medium,
the environmental condition during the incubation period and the level of
plant growth regulators (auxin, cytokinins, gibberellins, ethylene, etc.) is
a major factor that controls callus formation in the culture medium
(Evans, 2001).
     Establishment of a callus from the explants can be divided roughly
in to three developmental stages (Purohit et al., 2003).
1. During the initial induction phase metabolism is stimulated as the cells
  prepare for division. The length of this phase depends mainly on the
  physiological status of the explants cells as well as the culture

Chapter one                           Introduction and Literature Review

2. Subsequently, there is a phase of active cell division as the explants
  cells revert to a meristematic or "dedifferentiated" state.
3. A third phase involves the appearance of cellular differentiation and
  the expression of certain metabolic pathway that lead to the formation
  of secondary products (Yeoman, and Macleod, 1987). Interaction between Cytokinins and Auxins
    Auxins are a group of plant growth substances (often called plant
hormones or phytohormones); auxins play an essential role in
coordination of many growth and behavioral processes in the plant life;
on the cellular level, 'auxins' presence is essential for both cell division
and respective cell growth, resulting usually in its axial elongation (Zhao
et al., 2001).
    Cytokinins are a class of plant growth substances (plant hormones)
active in promoting cell division, and are also involved in cell growth,
differentiation, and other physiological processes; cytokinins are
generally promoting shoot development and inhibiting root development,
although they are necessary for cell division in both shoot and root apical
meristems (Bronner, 2004 ). In general, an increase in Auxin levels, such
as 2, 4-D led to decrease in the level of secondary metabolites (Kuang
and Cheng, 1981).
1.2.11. Strategies Adopted to increase the synthesis of secondary
   Cultured plant cells often produce reasonable quantities and different
profiles of secondary metabolites when compared with the intact plant;
the poor product expression in some cases attributed to the lack of
differentiation in cultures (Whitaker, 1997). On the other hand, there are
some cases that cultures over produce metabolites compared with the

Chapter one                            Introduction and Literature Review

whole plant (Kurz and Constabel, 1998). The choice of original plant
material having high-yields of the desired phytochemical may be
important in establishing high-yielding cultures (Deus and Zenk, 1982).
  Different strategies have been employed to increase secondary
metabolite production, of these that have yielded the best results are:- Culture Media
   Media components can play a vital role in stimulating the secondary
metabolite production, e.g. many plant cells that grown in limited amount
of nitrogen or phosphate gave enhanced yields of secondary metabolites
sucrose is the main carbon source of the most media; increasing the
sucrose concentration cause an increase in metabolites (Zenk, 1977).
   Cell Suspension cultures of Coleus blumei produce maximum yield of
rosmarinic acid (3.5 g/l) when 5% of sucrose was added to the culture
media but a sharp reduction up to (0.7 g/l) occurred in media containing
3% sucrose only (Zenk et al., 1977). Plant Growth Regulators
   Another important component of the culture media is the growth
regulator (phytohormone); auxins and cytokinins have shown the most
remarkable effects on growth and productivity of plant metabolites. In
general, an increase in auxin levels, such as 2, 4-D led to decrease in the
level of secondary metabolites (Kuang and Cheng, 1981). Staba, (1985)
reported that GA3 was also effective on plant cell cultures, the growth of
callus of dioxin-producing plant, Digitalis lanata, was promoted by the
addition of GA3 into the media. The suspension cultures of Catharanthus
roseus initiated from stem and leaf explants on a medium containing
NAA and kinetin had been established to increase secondary products
formation (Zhao et al., 2001).

Chapter one                          Introduction and Literature Review Physical Factors
    In vitro production of secondary metabolites by plant cell cultures is
largely dependent on environmental factors: light, pH, temperature,
oxygen and other factors (Zenk et al., 1977).
   Ramawat (2004) reported that blue light induced maximum
anthocyanin formation in Haplopappus gracilis cell suspension cultures,
whereas white light induced anthocyanin synthesis in Catharanthus
roseus and Populus spp.
   Plants are usually cultured in media having pH range of 5 to 6. The
pH of the growth medium can influence the production of phytochemicals
in cultured cells. Cultures of Daucus carota produced less anthocyanin
when grown at pH 5.5 than those grown at pH 4.5, since anthocyanin
content decreased by 90% at pH 5.5 compared to tissues grown at 4.5
(Sateesh, 2003).
1.2.12. Secondary Product Expression in vitro
   The production of secondary metabolites in vitro is possible through
plant tissue culture (Barnum, 2003). Secondary products such as vivid
pigments, aromatic compounds, flavors, and bioactive phytochemicals
can now be successfully accumulated in many plant in vitro cultures; in
vitro production of valuable plant secondary products (phytochemicals)
has become an industrially promising alternative to synthetic compounds
(Mary Ann L. Smith , 2000). More consistent product quality and yield,
continuous and homogenous supply of plant material in a uniform
physiological state, use of recombinant DNA technologies for yield
improvement, production of novel compounds in vitro, which are absence
in the parent plant material and the ability for large scale cultivation of
cells and organs in bioreactors for easier and higher product recoveries

Chapter one                           Introduction and Literature Review

(Zafar and Datta, 1992).
1.2.13. Use of Cell Lines in Cytotoxicity Development
    Use of in vitro assay systems for the screening of potential anticancer
agents has been common practice almost since the beginnings of cancer
chemotherapy in 1964 (Wilson, 2000). The last decade has been an
enormous trend towards isolated cellular systems, primary cells in culture
and cell lines; these systems provide the desirable complexity of
structurally and functionally intact cells combined with excellent
experimental accessibility (Masters, 2000). They offer the unique
possibility to elucidate interaction with vital cellular function such as
metabolism, intercellular communication, signal transduction, growth and
death that were formerly difficult to address (Gebhardt, 2000). An
increasing pressure is being put for a more comprehensive adoption of in
vitro testing in safety evaluation of chemicals for cancer chemotherapy;
the impetus for this originates partly from financial consideration, since
in vitro testing has considerable economic advantages over in vivo testing
(Freshney, 2001). There is also an increasing realization of the limitation
of animal models in relation to human metabolism, as more and more
metabolic differences between species come to be identified; finally and
as importantly, there is the moral pressure to reduce animal
experimentation (Wilson, 2000). The main disadvantages of this
technique are the good level of expertise, instability of continuous cell
lines resulting from their unstable aneuploid chromosomal constitution,
and loss of differentiation properties of the cultured cells, which requires
stable markers for characterization (Freshney, 1994).

Chapter one                           Introduction and Literature Review

1.2.14. Apoptosis
   Apoptosis, or programmed cell death, is a normal component of the
development and health of multi cellular organisms (Saraste and Pulkki,
2000). This makes apoptosis distinct from another form of cell death
called necrosis in which uncontrolled cell death leads to lyses of cells,
inflammatory responses and, potentially, to serious health problems (Kerr
et al., 1972). Apoptosis, by contrast, is a process in which cells play an
active role in their own death (which is why apoptosis is often referred to
as cell suicide); upon receiving specific signals instructing the cells to
undergo    apoptosis   a   number     of   distinctive   biochemical    and
morphological changes occur in the cell (Saraste and Pulkki, 2000).
   Families of proteins known as caspases are typically activated in the
early stages of apoptosis. These proteins breakdown or cleave key
cellular substrates that are required for normal cellular function including
structural proteins in the cytoskeleton and nuclear proteins such as DNA
repair enzymes (McLaughlin et al., 2001). The caspases can also activate
other degradative enzymes such as DNase, which begin to cleave
theDNA in the nucleus. The result of these biochemical changes is
appearance of morphological changes in the cell (Dash, 2006).
   Cell death is an important variable in cancer development, cancer
prevention and cancer therapy (Zerban et al., 1994).
1.2.15. Cytogenetic Analysis
   The act of division is the accumulation of a series of events that have
occurred since the last time a cell divided, the period between two mitotic
divisions defines as somatic cell cycle (Taylor, 1999). The time from the
end of mitosis to the start the next is called interphase; the period of
actual division, corresponding to the visible mitosis is called M-phase

Chapter one                          Introduction and Literature Review

(Becher et al., 1984). In order to divide, a eukaryotic somatic cell must
double its mass and then divided its components equally between the two
daughter cells; doubling of size is a continuous process, resulting from
transcription and translation of the genes that code for the protein
constituting the particular cell phenotype
(Kubbies et al., 1985). By contrast reproductions of the genome occur
only during specific period of DNA synthesis; Mitosis of a somatic cell
generates two identical daughter cells, each bearing a diploid complement
of chromosomes (Dufrain, 1983). Inter phase is divided in to period that
are defined by reference to the timing of DNA synthesis, as summarized:-
   – Cells are released from mitosis in to G1 phase, during which RNAs
      and proteins are synthesized, but there is no DNA replication
      (Jumae et al., 1999).
   – The initiation of DNA replication marks the transition from G1
      phase to the period of S phase; S phase is defined as lasting until
      the entire DNA has been replicated. During S phase, the total
      content of DNA increases from the diploid value of 2n to the fully
      replicated value of 4n (Allen et al., 1997).
   – The period from the end of S phase until mitosis is called G2
      phase. During this period, the cell has two complete diploid sets
      of chromosome , S phase was so called as the synthetic period
      when DNA is replication , G1 and G2 standing for the two "Gaps"
      in the cell cycle when there is no DNA synthesis. Inter phase is
      divided in to the G1 , S , and G2 period, demarcation between one
      cell cycle and the next is provided by mitosis(M) (Lewin, 1997).


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