Mridula Nambiar

Chromosomal translocations in cancer

Genetic alterations in DNA can lead to cancer when it is present in proto-oncogenes, tumor suppressor genes, DNA repair genes etc. Examples of such alterations include deletions, inversions and chromosomal translocations. Among these rearrangements chromosomal translocations are considered as the primary cause for many cancers including lymphoma, leukemia and some solid tumors. Chromosomal translocations in certain cases can result either in the fusion of genes or in bringing genes close to enhancer or promoter elements, hence leading to their altered expression. Moreover, chromosomal translocations are used as diagnostic markers for cancer and its therapeutics. In the first part of this review, we summarize the well-studied chromosomal translocations in cancer. Although the mechanism of formation of most of these translocations is still unclear, in the second part we discuss the recent advances in this area of research.

How does DNA break during chromosomal translocations

Chromosomal translocations are one of the most common types of genetic rearrangements and are molecular signatures for many types of cancers. They are considered as primary causes for cancers, especially lymphoma and leukemia. Although many translocations have been reported in the last four decades, the mechanism by which chromosomes break during a translocation remains largely unknown. In this review, we summarize recent advances made in understanding the molecular mechanism of chromosomal translocations.

Methyl angolensate, a natural tetranortriterpenoid induces intrinsic apoptotic pathway in leukemic cells

Methyl angolensate (MA), a natural tetranortriterpenoid, purified from Soymida febrifuga is examined for the first time for its anticancer properties. We find that MA inhibits growth of T‐cell leukemia and chronic myelogenous leukemia cells in a time‐ and dose‐dependent manner. Accumulation of cells in the subG1 peak, annexin V binding and DNA fragmentation suggested induction of apoptosis. Besides, upregulation of BAD (proapoptotic) and downregulation of BCL2 (antiapoptotic) gene products further supported induction of apoptosis. Loss of mitochondrial membrane potential, activation of caspase 9, caspase 3, cleavage of PARP, downregulation of Ku70/80 and phosphorylation of MAP kinases suggested that MA could induce intrinsic pathway of apoptosis in leukemic cells.

A novel DNA intercalator, butylamino-pyrimido[4′,5′:4,5]selenolo(2,3-b)quinoline, induces cell cycle arrest and apoptosis in leukemic cells

SummaryDNA intercalators are one of the most commonly used chemotherapeutic agents. Novel intercalating compounds of pyrimido[4′,5′:4,5]selenolo(2,3-b)quinoline series having a butylamino or piperazino group at fourth position (BPSQ and PPSQ, respectively) are studied. Our results showed that BPSQ induced cytotoxicity whereas PPSQ was cytostatic. The cytotoxicity induced by BPSQ was concentration- and time-dependent. Cell cycle analysis and tritiated thymidine assay revealed that BPSQ affects the cell cycle progression by arresting at S phase. The absence of p-histone H3 and reduction in the levels of PCNA in the cells treated with BPSQ further confirmed the cell cycle arrest. Further, annexin V staining, DNA fragmentation, nuclear condensation and changes in the expression levels of BCL2/BAD confirmed the activation of apoptosis. Activation of caspase 8 and lack of cleavage of caspase 9, caspase 3 and PARP suggest the possibility of BPSQ triggering extrinsic pathway for induction of apoptosis, which is discussed. Hence, we have identified a novel compound which would have clinical relevance in cancer chemotherapeutics.

Potential G-quadruplex formation at breakpoint regions of chromosomal translocations in cancer may explain their fragility

Genetic alterations like point mutations, insertions, deletions, inversions and translocations are frequently found in cancers. Chromosomal translocations are one of the most common genomic aberrations associated with nearly all types of cancers especially leukemia and lymphoma. Recent studies have shown the role of non-B DNA structures in generation of translocations. In the present study, using various bioinformatic tools, we show the propensity of formation of different types of altered DNA structures near translocation breakpoint regions. In particular, we find close association between occurrence of G-quadruplex forming motifs and fragile regions in almost 70% of genes involved in rearrangements in lymphoid cancers. However, such an analysis did not provide any evidence for the occurrence of G-quadruplexes at the close vicinity of translocation breakpoint regions in nonlymphoid cancers. Overall, this study will help in the identification of novel non-B DNA targets that may be responsible for generation of chromosomal translocations in cancer.

Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells

Cancer cells are often associated with secondary chromosomal rearrangements, such as deletions, inversions, and translocations, which could be the consequence of unrepaired/misrepaired DNA double strand breaks (DSBs). Nonhomologous DNA end joining is one of the most common pathways to repair DSBs in higher eukaryotes. By using oligomeric DNA substrates mimicking various endogenous DSBs in a cell-free system, we studied end joining (EJ) in different cancer cell lines. We found that the efficiency of EJ varies among cancer cells; however, there was no remarkable difference in the mechanism and expression of EJ proteins. Interestingly, cancer cells with lower levels of EJ possessed elevated expression of BCL2 and vice versa. Removal of BCL2 by immunoprecipitation or protein fractionation led to elevated EJ. More importantly, we show that overexpression of BCL2 or the addition of purified BCL2 led to the down-regulation of EJ. Further, we found that BCL2 interacts with KU proteins both in vitro and in vivo. Hence, our results suggest that EJ in cancer cells could be negatively regulated by the anti-apoptotic protein, BCL2, and this may contribute toward increased chromosomal abnormalities in cancer.

Mechanism of Fragility at BCL2 Gene Minor Breakpoint Cluster Region during t(14;18) Chromosomal Translocation * □

Background: The mechanism of fragility at mcr during t(14;18) translocation is not known. Results: RAGs nick mcr using a unique mechanism involving the CCACCTCT motif, which is critical for its fragility. Importantly, mcr undergoes synapsis with RSS within cells, which is RAG-dependent. Conclusion: RAGs are responsible for fragility at mcr. Significance: The mechanism identified herein may help in understanding how DNA breaks during other translocations. The t(14;18) translocation in follicular lymphoma is one of the most common chromosomal translocations. Breaks in chromosome 18 are localized at the 3′-UTR of BCL2 gene or downstream and are mainly clustered in either the major breakpoint region or the minor breakpoint cluster region (mcr). The recombination activating gene (RAG) complex induces breaks at IgH locus of chromosome 14, whereas the mechanism of fragility at BCL2 mcr remains unclear. Here, for the first time, we show that RAGs can nick mcr; however, the mechanism is unique. Three independent nicks of equal efficiency are generated, when both Mg2+ and Mn2+ are present, unlike a single nick during V(D)J recombination. Further, we demonstrate that RAG binding and nicking at the mcr are independent of nonamer, whereas a CCACCTCT motif plays a critical role in its fragility, as shown by sequential mutagenesis. More importantly, we recapitulate the BCL2 mcr translocation and find that mcr can undergo synapsis with a standard recombination signal sequence within the cells, in a RAG-dependent manner. Further, mutation to the CCACCTCT motif abolishes recombination within the cells, indicating its vital role. Hence, our data suggest a novel, physiologically relevant, nonamer-independent mechanism of RAG nicking at mcr, which may be important for generation of chromosomal translocations in humans.

G-quadruplex structures formed at the HOX11 breakpoint region contribute to its fragility during t(10;14) translocation in T-cell leukemia.

ABSTRACT The t(10;14) translocation involving the HOX11 gene is found in several T-cell leukemia patients. Previous efforts to determine the causes of HOX11 fragility were not successful. The role of non-B DNA structures is increasingly becoming an important cause of genomic instability. In the present study, bioinformatics analysis revealed two G-quadruplex-forming motifs at the HOX11 breakpoint cluster. Gel shift assays showed formation of both intra- and intermolecular G-quadruplexes, the latter being more predominant. The structure formation was dependent on four stretches of guanines, as revealed by mutagenesis. Circular dichroism analysis identified parallel conformations for both quadruplexes. The non-B DNA structure could block polymerization during replication on a plasmid, resulting in consistent K+-dependent pause sites, which were abolished upon mutation of G-motifs, thereby demonstrating the role of the stretches of guanines even on double-stranded DNA. Extrachromosomal assays showed that the G-quadruplex motifs could block transcription, leading to reduced expression of green fluorescent protein (GFP) within cells. More importantly, sodium bisulfite modification assay showed the single-stranded character at regions I and II of HOX11 in the genome. Thus, our findings suggest the occurrence of G-quadruplex structures at the HOX11 breakpoint region, which could explain its fragility during the t(10;14) translocation.

A novel structural derivative of natural alkaloid ellipticine, MDPSQ, induces necrosis in leukemic cells.

SummaryDNA intercalating molecules are promising chemotherapeutic agents. In the present study, a novel DNA intercalating compound of pyrimido[4′,5′:4,5]selenolo(2,3-b)quinoline series having 8-methyl-4-(3 diethylaminopropylamino) side chain is studied for its chemotherapeutic properties. Our results showed that 8-methyl-4-(3 diethylaminopropylamino) pyrimido [4′,5′:4,5] selenolo(2,3-b)quinoline (MDPSQ) induces cytotoxicity in a time- and concentration-dependent manner on leukemic cell lines. Both cell cycle analysis and tritiated thymidine assays revealed that MDPSQ affects DNA replication. Treatment with MDPSQ resulted in both elevated levels of DNA strand breaks and repair proteins, further indicating its cytotoxic effects. Besides, Annexin V/PI staining revealed that MDPSQ induces cell death by triggering necrosis rather than apoptosis.

Prevalence and analysis of t(14;18) and t(11;14) chromosomal translocations in healthy Indian population

Hematopoietic malignancies like leukemia and lymphoma are characteristically associated with various chromosomal translocations. Follicular lymphoma (FL) and mantle cell lymphoma (MCL) are two subtypes of non-Hodgkin’s lymphoma which possess t(14;18) and t(11;14) translocations, respectively. The incidence of FL and MCL is higher in the western countries as compared to India. Interestingly, the associated translocations are also found in healthy individuals in western population, which is 50–80% for t(14;18), whereas t(11;14) occurs at a very low frequency. However, there are no studies to explore these translocations in healthy Indian population, which could explain the lower incidence of FL and MCL. We employed Southern hybridization following nested PCR to detect above translocations in healthy individuals from India. Our results suggest that this assay can detect one t(14;18) translocation event in up to 107 normal cells where as one t(11;14) in 108 normal cells. According to our results, 87 out of 253 individuals carry t(14;18) indicating 34% prevalence in the population. The presence of this translocation was also detectable at the transcript level. Although, no gender-based difference was observed, an age-dependent increase in the prevalence of translocation was found in adults. However, even after studying 210 people, we could not detect any t(11;14) translocation, indicating that it is uncommon in Indian population. These results suggest that lower incidence of FL and MCL in India could be attributed to lower prevalence of these translocations in healthy individuals.