PI: Professor Indraneel Mittra

Lab Members


Primary research interest:

Biology of extracellular nucleic acids and their role in cancer, ageing and other diverse pathologies.

Research projects

  1. Genomic integration of circulating nucleic acids and DNA damage.
Whether nucleic acids that circulate in blood (CNAs) have any patho-physiological functions in the host have not been explored. Our lab has demonstrated for the first time that CNAs in the form of fragmented DNA and chromatin (DNAfs and Cfs) can freely enter into healthy cells, associate with their chromosomes and integrate into host cell genomes. The latter leads to activation of DDR and up-regulation of apoptotic pathways. When injected intravenously into mice, DNAfs and Cfs integrate into cells of vital organs and bring about dsDNA breaks and apoptosis in a proportion of cells.

These findings lead to the conclusion that circulating DNAfs and Cfs are physiological, continuously arising, endogenous DNA damaging agents with implications for ageing and a multitude of human pathologies including initiation of cancer. All the pathological actions of DNAfs and Cfs described above can be abrogated by concurrent treatment with DNase I and / or anti-histone antibody complexed nanoparticles both in vitro and in vivo suggesting therapeutic possibilities.

Genomic integration of Cfs involves DNA double strand break repair. Mice were injected i.v. with Cfs (100 ng DNA) and sacrificed 24 hr later. Sections of brain were processed for immuno-FISH using human-specific genomic probe (green) and antibodies against g-H2AX (red). Co-localization of green and red signals are clearly visible.

  1. Chromatin isolated from serum of cancer patients trigger genomic instability, inflammation and cancer.
Our research has shown that Cfs derived from cancer patients are biologically more potent than those derived from healthy volunteers. We have observed that treatment of NIH3T3 cells with Cfs isolated from sera of cancer patients trigger a genome-wide deregulation of transcription, chromosomal instability, inflammation and up-regulation of multiple cancer-related pathways encompassing 200 genes. In 30% of our experiments, oncogenic transformation was observed in the treated cells within 72hr - 96hr. The transformed cells were tumourigenic in immune-deficient mice and FISH detected human DNA signals in nuclei of tumour cells. Cfs that circulate in blood of cancer patients may be involved in systemic spread of cancer.

Induction of chromosomal instability following treatment of recipient cells with Cfs derived from sera of cancer patients as detected by spectral karyotyping. The treated cells show significantly increased number of chromosomal translocations. It should be noted that the treated cell shows evidence of hyper-diploidy

  1. Chromatin from dead cancer cells integrates into genomes of living cells to induce DNA damage, inflammation, chromosomal instability and cancer.
Following our observation that circulating Cfs are DNA damaging and oncogenic agents, we investigated whether application of dead cancerous cells (dcCfs) to NIH3T3 cells will bring about similar pathological changes. We found that numerous dcCfs exiting from the dead cells rapidly entered into the recipients, integrated into their genomes and triggered genome-wide de-regulation of transcription. Whole genome sequencing of NIH3T3 cells treated with dead human cancer cells detected several hundred thousand human reads in recipient mouse cells. Microarray analysis revealed up-regulation of multiple pathways related to phagocytosis, inflammation and cancer. Intracellular dcCfs induced dsDNA breaks, chromosomal instability, activation of apoptotic pathways and an intense up-regulation of inflammatory cytokines. Treatment with some types of dead cancerous cells induced rapid oncogenic transformation of NIH3T3 cells within 72hr - 96 hr; the transformed cells were tumourigenic in immune-deficient mice and FISH detected presence of human DNA signals in nuclei of tumour cells. Treatment with some of the other types of dead cancer cells led to senescence of the recipients. Intravenous injection of dead human cancer cells into mice activated H2AX, Caspase-3 and inflammation in cells of distant organs while FISH detected human DNA signals in their nuclei. Our results suggest that dead cancer cells may be responsible for local spread of cancer via the medium of dcCfs and may initiate new cancers in cells of distant organs if dead cancer cells were to enter into the blood stream.

Nuclear uptake of BrdU labeled dcCfs is prevented by concurrent treatment with CNPs.

  1. How histologically similar is the metastatic tumour to the primary?
There is a long held belief among medical doctors and scientists that metastatic tumours are histologically similar to the primary. This belief is reified because it supports the prevailing theory of cancer metastasis. The latter teaches that tumour cells detach from the primary site to intravasate into the blood stream reach distant organs where they extravasate into the target tissues and grow to form a metastatic cancer. However, there are several problems with the prevailing theory which have remained unexplained. Our research findings have led to an alternative theory that proposes that metastases are new cancers initiated in cells of target organs through a process of de novo oncogenesis by fragmented chromatin derived from dead cancer cells.

To test this hypothesis we have taken two approaches. In the first, we have collected pathological slides of 400 metastatic tumours from different sites. Three senior pathologists will be asked to identify the primary sites from where these metastases arose without prior knowledge of their primary origin. This study will scientifically test for the first time the widely held belief that metastatic tumours are histologically similar to the primary. In the second approach, we have taken nearly 300 primary and metastatic tumours of diverse origins. If our alternative theory of metastasis were to be true that metastatic tumours arise de novo from cells of target organs rather than primary tumours growing at distant sites, the metastatic tumours should express at least some of the normal antigens of visceral cells from which they arise. The primary tumours, on the other hand, should not express these antigens.

  1. Pullulan-histone antibody nanoconjugates for the removal of chromatin fragments from systemic circulation.
Since our study shows that Cfs are DNA damaging agents with implications for diverse diseases associated with elevated Cfs levels, removal of Cfs could be of therapeutic value. We have developed pullulan-based histone antibody nanoconjugates for the removal of Cfs from circulation. Nanoconjugates were developed and various physico-chemical characterizations were carried out. The efficacy of these nanoconjugates on removing Cfs was evaluated both in vitro and in vivo. Our results indicate that nanoconjugates may have therapeutic value in reducing inflammation and fatality in a mouse model of sepsis and in preventing neutropenia following treatment with Adriamycin both of which are mediated via Cfs.

  1. Chromatin from dead cells cause radiation induced by-stander effect.
Radiation induced by-stander effect (RIBE) is a poorly understood phenomenon wherein non-irradiated cells show evidence of DNA damage, genomic instability, mutation and apoptosis both in vitro and in vivo. We have observed that chromatin fragments (Cfs) that are released from dead cells resulting from radiation treatment are one of the key factors responsible for RIBE. We employed the widely used trans-well plate system wherein NIH3T3 mouse fibroblast cells grown on trans-well filters were irradiated using a Co60 g-ray source, and RIBE in the form of dsDNA breaks represented by H2AX activation were analyzed in recipient NIH3T3 cells grown on tissue culture plates. gH2AX signals increased approximately 3-fold following 6hr incubation of irradiated cells with the recipient cells confirming activation of RIBE. However, the number of gH2AX signals dropped dramatically when donor cells were treated at the time of irradiation with anti-histone antibody complexed nanoparticles (CNPs), which are known to specifically bind to and inactivate Cfs. Since blood levels of Cfs increase following radiotherapy, Cfs may induce RIBE in cells of various organs of the body and be responsible for side-effects of radiotherapy for cancer.

  1. A paradoxical synergistic effect between Resveratrol and copper (II) with respect to degradation of DNA and RNA.
Since our study shows that Cfs are DNA damaging agents with implications for diseases associated with elevated Cfs levels, removal / degradation of Cfs could be of therapeutic value. Resveratrol (R), a plant polyphenol, is known to reduce Cu (II) to Cu (I) generating reactive oxygen species that can cleave plasmid DNA. We have observed that there exists a synergistic effect between R and Cu whereby plasmid DNA cleaving / degrading activity of R-Cu progressively increases as the ratio of R to Cu is increased i.e., the concentration of Cu is successively reduced with respect to a fixed concentration R. Whereas cleavage of DNA occurs at low molar ratios of R to Cu, at higher ratios, complete degradation of DNA is achieved. With further increases in the ratio, wherein the concentration of Cu is reduced to very low levels, the DNA degrading activity of R-Cu is gradually lost. This synergistic effect is also seen with respect to eukaryotic genomic DNA and RNA. This data indicate that R-Cu may have potential in vivo Cfs degrading activity and suggest that it might be possible to reduce the concentration of Cu to our advantage so as to obviate any toxic side-effects of Cu.

Increasing cleavage / degradation of plasmid DNA by R-Cu in the presence of decreasing concentrations of Cu.


  1. The ability of R-Cu to degrade circulating chromatin in vivo.
Following cue from the above in vitro experiments, we undertook in vivo studies of R-Cu to test its Cfs-degrading activity. We observed that  R-Cu at very high molar ratios of R to Cu, i.e., at low concentration of Cu with respect to R, can 1) prevent LPS- and Adriamycin-induced Cfs surge in mice; 2) prevent lethality of mice challenged with LPS; 3) prevent DNA damaging effects of Adriamycin in visceral organs and prevention of neutropenia; 4) prevent lung metastasis in the B16-F10 mouse melanoma model.

  1. Sonication imparts biological properties to DNA and ability to transmit itself horizontally across species and taxonomical kingdoms.
We have recently reported that DNA and chromatin fragments derived from apoptotic cells that circulate in blood of human beings can readily enter into somatic cells of mice in vitro and in vivo, evoke a DNA damage repair response and integrate themselves into their genomes. However, these findings are at odds with established knowledge on two counts: first, DNA is not known to spontaneously enter into cells, and second, DNA is not known to have any intrinsic biological properties. We hypothesized that cellular entry and acquisition of biological properties are functions of the size of DNA. To test this hypothesis we isolated HMW DNA from cancerous and non-cancerous human cells, bacteria and plant and sonicated them to generate fragments similar in size to circulating DNA. Our early results show that while HMW DNAs are incapable of entering into mouse cells, sonicated DNA (sDNA) from human, bacteria and plant sources could do so spontaneously. Likewise, human, bacterial and plant sDNA could spontaneously enter into bacterial cells while their HMW counterparts could do so sparingly. The intracellular sDNA associated themselves with host cell chromosomes and integrated themselves into their genomes. We also demonstrate that sDNA, but not HMW DNA, from human, bacterial and plant sources can phosphorylate H2AX and activate the transcription factor NFkB in mouse cells, indicating that sDNAs have acquired biological properties. Taken together, our findings might provide a mechanistic explanation for reported evidence of horizontal transfer of genes in nature and suggest that extreme environmental stress leading to cellular apoptosis and DNA fragmentation may have played a significant role in adaptation and evolution of species.

Nuclear uptake of BrdU-labelled bacterial s-DNA by NIH3T3 cells.