HYPOTHESIS broad spectrum of malignanciesVS1 in early

HYPOTHESIS AND
SPECIFIC AIMS

Defects
in DNA repair genes is often associated with increased risk of developing
certain types of cancer. However mutations in one such gene that codes for a
DNA helicase BLM, predisposes the affected individuals to a broad spectrum of malignanciesVS1  in early life1.Blooms syndrome (BS) is a rare disorder that affects
multiple systems in the body and is associated with growth deficits,
radiosensitivity, compromised immune system, insulin resistance,
neurodegeneration, cancer predisposition and aging2. Previous studies have established that BS cells are
associated with cellular redox alterations and they exist in prooxidant state in vivoVS2 3,
4, 5.Theoretically,
it has also been suggested that BS is an excellent candidate of mitochondrial
alterations since mitochondria is the main endogenous ROS generating source6.However no studies
conducted till date to validate the sameVS3 . Oxidative stress and impairment of
mitochondrial homeostasis provide a more plausible explanation for the
multifaceted phenotypes that is observed in patients with BS7.

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A
preliminary screen conducted in the lab identified a mitochondrial protein,
TFAM to be significantly upregulated in cells isolated from BS patient. TFAM
(Mitochondrial Transcription Factor A) is a nuclear encoded mitochondrial DNA
transcription factor that regulates transcription, replication of mitochondrial
DNA and mitochondrial copy number8.
The central
hypothesis of this project is that mitochondrial dysfunction (MDF) and
oxidativeVS4  stress (OS) contributes to some of
the phenotypes (genome instability, sister chromatid exchanges, cancer
predisposition) that is observed in patients with Bloom syndrome. The rationale
behind this hypothesis is that OS and MDF have been described as a hallmark of
many cancer prone syndromes9.
Our long-term goal is to dissect the molecular mechanism
linking OS and MDF to pathogenesisVS5  of Bloom
syndrome; as this will aid in the development of clinical strategies focussed
on mitigating disease progression in affected patients. The following specific aims will test
the central hypothesis.

  Aim1:
To characterize the oxidative stress
phenotype observed in Bloom’s syndromeVS6 

 

a.   
To investigate the oxidant-antioxidant
status of BLM knockout (KO) cells and to compare antioxidant enzyme activities
with wildtype (WT) cells.
Western Blotting and qPCR will be performed to analyse the levels of oxidative
stress biomarkers and the functionality of antioxidants will be examined
through enzyme activity assays

 

b.   
To characterize the susceptibility of
BLM KO cells to oxidative injury using
clonogenic assay.

 

The above experiments will determine
whether the oxidative stress phenotype observed in patients with BS is due to
overproduction of ROS or impairment of antioxidant defenses.

 

Aim2: To
investigate and understand mitochondrial alterations in BS

 

a.    To
investigate the effect of TFAM upregulation by determining whether an increase in TFAM corresponds
to more mitochondria by using fluorescence microscopy, flow cytometry and qPCR.

 

b.    To
investigate the cause of TFAM upregulation by performing Western Blotting, qPCR and
Immunofluorescence to study levels of TFAM and its transcription factors

 

c.    To
perform biochemical characterization of mitochondrial function by analyzing
mitochondrial features
like mitochondrial membrane potential, respiratory function, mROS production,
expression of mtDNA repair proteins etc. to detect mitochondrial dysfunction by
using a combination of fluorescence microscopy, Agilent Seahorse XF Cell Mito
Stress Test and Western Blotting.

 

This
will experimentally validate the presence of mitochondrial abnormalities in
Blooms syndrome

 

Aim3: To determine
the role of oxidative stress in pathophysiology of BS

 

a.   
To understand the molecular mechanism
through which OS contributes to phenotypes observed in Blooms syndrome by determining whether OS directly
contributes to genome instability or whether it impairs the function of DNA
repair proteins. This will be done using a combination of microscopy, DNA
damage and repair kits.

 

b.   
To study whether the supplementation
of ROS scavengers/antioxidants (esp targeted to mitochondria) improve/delay
some of the symptoms of BS like
sister chromatid exchanges, sensitivity to damaging agents, cellular and
mitochondrial ROS levels using microscopy and viability assays.

 

This
will determine whether oxidative stress elicits BS phenotypes and whether it
can be further exploited for therapeutic applications

 

Dissecting
the role of OS and MDFVS7  in pathogenesis of BS will have a
broad impact on the field by providing insights into pathogenesis of other
cancer-prone syndromesVS8 . A better understanding of this
mechanism will aid in the development of therapeutic strategiesVS9  that will alleviate some of the
phenotypes (ageing and tumor development) of patients suffering from the
disease.

 

 VS1Hook

 

 VS2What
is known?

 VS3Gap
in knowledge

 VS4Central
hypothesis

 VS5overview

 VS6Aim
title

 VS7OUTCOME

 VS8IMPACT

 VS9INNOVATION