Zukile Mbita

De-regulation of the RBBP6 isoform 3/DWNN in human cancers

Retinoblastoma binding protein 6 (RBBP6) is a nuclear protein, previously implicated in the regulation of cell cycle and apoptosis. The human RBBP6 gene codes for three protein isoforms and isoform 3 consists of the domain with no name domain only whilst the other two isoforms, 1 and 2 comprise of additional zinc, RING, retinoblastoma and p53 binding domains. In this study, the localization of RBBP6 using RBBP6 variant 3 mRNA-specific probe was performed to investigate the expression levels of the gene in different tumours and find a link between RBBP6 and human carcinogenesis. Using FISH, real-time PCR and Western blotting analysis our results show that RBBP6 isoform 3 is down-regulated in human cancers. RBBP6 isoform 3 knock-down resulted in reduced G2/M cell cycle arrest whilst its over-expression resulted in increased G2/M cell cycle arrest using propidium iodide DNA staining. The results further demonstrate that the RBBP6 isoform 3 may be the cell cycle regulator and involved in mitotic apoptosis not the isoform 1 as previously reported for mice. In conclusion, these findings suggest that RBBP6 isoform 3 is a cell cycle regulator and may be de-regulated in carcinogenesis.

Human immunodeficiency virus-1 (HIV-1)-mediated apoptosis: new therapeutic targets.

HIV has posed a significant challenge due to the ability of the virus to both impair and evade the host’s immune system. One of the most important mechanisms it has employed to do so is the modulation of the host’s native apoptotic pathways and mechanisms. Viral proteins alter normal apoptotic signaling resulting in increased viral load and the formation of viral reservoirs which ultimately increase infectivity. Both the host’s pro- and anti-apoptotic responses are regulated by the interactions of viral proteins with cell surface receptors or apoptotic pathway components. This dynamic has led to the development of therapies aimed at altering the ability of the virus to modulate apoptotic pathways. These therapies are aimed at preventing or inhibiting viral infection, or treating viral associated pathologies. These drugs target both the viral proteins and the apoptotic pathways of the host. This review will examine the cell types targeted by HIV, the surface receptors exploited by the virus and the mechanisms whereby HIV encoded proteins influence the apoptotic pathways. The viral manipulation of the hosts’ cell type to evade the immune system, establish viral reservoirs and enhance viral proliferation will be reviewed. The pathologies associated with the ability of HIV to alter apoptotic signaling and the drugs and therapies currently under development that target the ability of apoptotic signaling within HIV infection will also be discussed.

Can targeting apoptosis resolve the cancer saga

A defect in apoptosis is almost always linked to many pathologies, including cancer. Carcinogenesis has been linked to abnormalities in the apoptotic pathway, and many drugs that are targeted at different parts of this pathway are being developed. There have been many promising drugs that target the extrinsic death receptor pathway as well as the intrinsic mitochondrial apoptotic pathway. There have also been developments in targeting initiator and effector caspases, as well as the death domains that are involved in transducing the apoptotic signals. In this review, the authors will briefly explain how apoptosis deregulation can lead to cancer and discuss drugs that promise success in targeting this anomaly. This article shall also explain how co-treatments with chemotherapy can increase survival of cancer patients. There is a problem of acquired resistance in some of these therapies but there may be ways to overcome this.

The Role of MicroRNAs in Kidney Disease

MicroRNAs (miRNAs) are short noncoding RNAs that regulate pathophysiological processes that suppress gene expression by binding to messenger RNAs. These biomolecules can be used to study gene regulation and protein expression, which will allow better understanding of many biological processes such as cell cycle progression and apoptosis that control the fate of cells. Several pathways have also been implicated to be involved in kidney diseases such as Transforming Growth Factor-β, Mitogen-Activated Protein Kinase signaling, and Wnt signaling pathways. The discovery of miRNAs has provided new insights into kidney pathologies and may provide new innovative and effective therapeutic strategies. Research has demonstrated the role of miRNAs in a variety of kidney diseases including renal cell carcinoma, diabetic nephropathy, nephritic syndrome, renal fibrosis, lupus nephritis and acute pyelonephritis. MiRNAs are implicated as playing a role in these diseases due to their role in apoptosis, cell proliferation, differentiation and development. As miRNAs have been detected in a stable condition in different biological fluids, they have the potential to be tools to study the pathogenesis of human diseases with a great potential to be used in disease prognosis and diagnosis. The purpose of this review is to examine the role of miRNA in kidney disease.

Abstract 5546: Screening the RbBP6 gene for abnormal alternative splicing in colon cancer

Cancer arises after a series of mutations that result in uncontrolled cellular differentiation and growth. These result because the genes required for normal cell growth and apoptotic processes are abnormally regulated and thus the progression of cancer and survival is favoured. The RbBP6 gene is amongst the genes which are down-regulated in cancerous cells. RbBP6 is a gene that has been shown to regulate cell cycle control and apoptosis. RbBP6 isoform 3 has been shown to regulate G2/M cell cycle checkpoint while the alternatively spliced RbBP6 transcript 1 product has been implicated in apoptosis regulation and cancer. Expression and regulation of the gene in colon cancer was examined by amplifying cDNA of RNA extracted from three colon adenocarcinoma cell lines obtained from different stages of colon cancer, using RT-PCR with the RbBP6 exon 16 primers and confirmation of the 2 splice variants by NCBI BLAST. Two bands appeared on a 1% Agarose gel, the ratio of the intensity of the two bands was found to be 1:1 in the primary cell line compared to down-regulation in grade II cell line while these alternatively spliced products could hardly be amplified in grade III cell line. RbBP6 has two splice isoforms which are regulated by alternative splicing in colon cancer. The expression of these splice variants was found to be indirectly proportional to colon cancer progression. The primary colon cancer cell line showed more expression of these alternatively spliced products, which decreased significantly in grade II and III. This suggests that RbBP6 alternatively spliced products could be anti-carcinogenic but are de-regulated in favour of carcinogenesis and this further suggests that RbBP6 gene might be somehow involved in the development and progression of colon adenocarcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5546. doi:10.1158/1538-7445.AM2011-5546

Translocator Protein (TSPO) as a Potential Biomarker in Human Cancers

TSPO is a receptor involved in the regulation of cellular proliferation, apoptosis and mitochondrial functions. Previous studies showed that the expression of TSPO protein correlated positively with tumour malignancy and negatively with patient survival. The aim of this study was to determine the transcription of Tspo mRNA in various types of normal and cancer tissues. In situ hybridization was performed to localise the Tspo mRNA in various human normal and cancer tissues. The relative level of Tspo mRNA was quantified using fluorescent intensity and visual estimation of colorimetric staining. RT-PCR was used to confirm these mRNA levels in normal lung, lung cancer, liver cancer, and cervical cancer cell lines. There was a significant increase in the level of transcription in liver, prostate, kidney, and brain cancers while a significant decrease was observed in cancers of the colon and lung. Quantitative RT-PCR confirmed that the mRNA levels of Tspo are higher in a normal lung cell line than in a lung cancer cell line. An increase in the expression levels of Tspo mRNA is not necessarily a good diagnostic biomarker in most cancers with changes not being large enough to be significantly different when detected by in situ hybridisation.

Abnormalities in alternative splicing of angiogenesis-related genes and their role in HIV-related cancers

Alternative splicing of mRNA leads to an increase in proteome biodiversity by allowing the generation of multiple mRNAs, coding for multiple protein isoforms of various structural and functional properties from a single primary pre-mRNA transcript. The protein isoforms produced are tightly regulated in normal development but are mostly deregulated in various cancers. In HIV-infected individuals with AIDS, there is an increase in aberrant alternative splicing, resulting in an increase in HIV/AIDS-related cancers, such as Kaposi’s sarcoma, non-Hodgkin’s lymphoma, and cervical cancer. This aberrant splicing leads to abnormal production of protein and is caused by mutations in cis-acting elements or trans-acting factors in angiogenesis-related genes. Restoring the normal regulation of alternative splicing of angiogenic genes would alter the expression of protein isoforms and may confer normal cell physiology in patients with these cancers. This review highlights the abnormalities in alternative splicing of angiogenesis-related genes and their implication in HIV/AIDS-related cancers. This allows us to gain an insight into the pathogenesis of HIV/AIDS-related cancer and in turn elucidate the therapeutic potential of alternatively spliced genes in HIV/AIDS-related malignancies.

Significant up-regulation of 1-ACBP, B-ACBP and PBR genes in immune cells within the oesophageal malignant tissue and a possible link in carcinogenic angiogenesis.

Oesophageal cancer ranks as the sixth most common malignancy in the world, and recent evidence has shown that its incidence is increasing. ACBPs (Acyl-coA binding proteins) act as intracellular carrier-proteins for medium to long chain acyl-coA, mediating fatty acid transport to the mitochondrion for ß-oxidation. ACBPs are also believed to be putative ligands of PBR (peripheral benzodiazepine receptor), and once they bind to this receptor they facilitate mitochondrial membrane permeabilization, presumably favouring apoptosis. The main aim of the study was to establish the expression patterns of 1- Acyl-coA binding proteins (1-ACBP), B- Acyl-coA binding proteins (B-ACBP), and peripheral bezodiazepine receptor (PBR) in oesophageal cancer, and to link their roles with the disease. In situ hybridization and quantitative real-time PCR methods were performed to determine localization and the expression levels of the three genes in oesophageal cancer. All three genes illustrated substantial up-regulation within the malignant tissue sections as compared to normal oesophageal sections, all three transcripts localized specifically to mast cells, plasma cells and lymphocytes in diseased and normal tissue section. In the diseased tissue B-ACBP and 1-ACBP mRNA localized to endothelial cells of blood vessels in the submucosa. B-ACBP also localized to the nucleus of squamous epithelial cells. PBR localization was indicated in tumour islands of invasive tissue sections. Quantitative RT-PCR also indicated that the expression levels of PBR were higher as compared to the ACBP genes expression in tumours. These results show that 1-ACBP, B-ACBP and PBR play a role in the pathogenesis of oesophageal tumours and possibly in carcinogenic angiogenesis.

Abstract 1928: Characterization of the RPL9 and LIAS genes in lung tumors.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC RPL9 is located on chromosome 4p14 and is approximately 5.5 kb in length and contains 8 exons. The message for human RPL9 is 712 nucleotides long. Some of the functions of RPL9 documented this far include the crucial involvement of the gene product in cell proliferation and protein biosynthesis. Lipoic acid synthetase (LIAS) is a 1.73 kb gene located at chromosome 4p14. Alternative splicing occurs at this locus and two transcript variants encoding distinct isoforms have been identified. The protein encoded by LIAS gene belongs to the biotin and lipoic acid synthetases family and localizes in the mitochondrion. The main objective of this study was to evaluate the expression pattern of RPL9 and LIAS in lung cancer and to characterise their role in apoptosis and also to determine if the expression pattern of this genes varies between normal and diseased state of the tissue. In Situ hybridization, quantitative Real Time PCR, TUNEL and Bio-informatics have been employed in order to attain the objectives of this study. In Situ hybridization showed that RPL9 localises in the cytoplasm and it is up-regulated in lung cancer relative to normal lung. LIAS localises in the cytoplasm and it is also up-regulated in lung cancer. The expression of RPL9 was relatively higher than that of LIAS determined by the intensity of localisation. Quantitative real time PCR confirmed the up-regulation of RPL9 and LIAS in lung cancer. RPL9 and LIAS were found to be up-regulated 8 and 4 fold respectively in lung A549 lung adenocarcinoma relative to MRC5 normal lung fibroblast cell lines. TUNEL showed the highest DNA fragmentation in adenocarcinoma, followed by squamous cell lung carcinoma then large cell lung carcinoma which is the same pattern observed in RPL9 and LIAS mRNA localisation by In Situ hybridization. To further characterise the role of RPL9 and LIAS in human, Bio-informatics tools were used and the results revealed that RPL9 is highly conserved through evolution, up-to 100 % identical to chimpanzee and 98 % to mouse. LIAS was found to be 91 % identical to rat and 90 % identical to mouse. All these discoveries coupled with resistance to apoptosis of CHO cell line in which RPL9 and LIAS were found to be mutated following promoter-trap mutagenesis, strongly suggests that RPL9 might be playing a role in cell cycle and apoptosis. RPL9 has been highly conserved through evolution. Manipulation of this gene can lead to greater biological discoveries in cancer research and the elevated expression of RPL9 can be used as a molecular marker for early detection of cancer Citation Format: Zodwa Dlamini, Zukile Mbita, Lebogang Mphahlele. Characterization of the RPL9 and LIAS genes in lung tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1928. doi:10.1158/1538-7445.AM2013-1928 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 99: Posttranslational modification of the RBBP6 isoform 3 is required for excessive cell proliferation

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL RBBP6 isoform 3 belongs to a family of RBBP6 cell cycle-related proteins. There are 3 human RBBP6 isoforms; iso 1, 2 and 3, transcribed from RBBP6 mRNA variants 1, 2 and 3 respectively. RBBP6 isoform 1 has been shown to be a pro-apoptotic protein while the function of isoform 2 has not been confirmed. Recently isoform 3 has shown to be involved in G2/M cell cycle regulation and is deregulated in human cancers. This study aimed at investigating how the RBBP6 isoform 3 regulates cell proliferation. Cell culture and transfections were used to determine the effect of RBBP6 isoform 3 on cell cycle progression. RBBP6 variant 3 mRNA was cloned into a pCDNA3.1/Zeo (+) vector and expressed in human kidney embryonic cells, Hek 293T. Cell cycle and cell counts analysis were conducted by using Flow Assorted Cell Sorting (FACS). Western blotting analysis was used to analyse RBBP6 isoform 3 using a polyclonal antibody against RBBP6 isoform 3 N-terminal region. Transfection of Hek 293T cells with RBBP6 variant 3 mRNA resulted in temporal G2/M cell cycle arrest. This cell cycle arrest ceased after 48 hours and Western blotting showed that RBBP6 isoform 3 was posttranslationally modified through either glycosylation or phosphorylation or both. Mass spectrometry confirmed that the higher molecular weight protein was indeed RBBP6 isoform 3. This higher molecular weight product was accompanied by excessive cell proliferation. This result suggests that cancer cells use this posttranslational modification to bypass G2/M cell cycle arrest. RBBP6 isoform 3 could therefore be targeted for new anti-cancer therapeutic strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 99. doi:1538-7445.AM2012-99