Cancer Research in Action Conference Poster Awards
Posters displayed at CANSA’s Cancer Research in Action Conference
A total of 21 posters were submitted by students and researchers from academic institutions throughout South Africa. All were of an exceptionally high standard, 7 posters were awarded prizes at the gala dinner evening.
HIV INFECTION IS ASSOCIATED WITH HIGH PERCENTAGE OF MEMORY CD4 AND CD8 LYMPHOCYTES IN A POPULATION OF WOMEN WITH CERVICAL CANCER IN KWAZULU-NATAL, SOUTH AFRICA.
Sebastian D, Moodley M, Assounga Agh.
Background: Memory T lymphocytes play a major role in the immune response. Is the ratio of memory to naïve T lymphocytes between CD4 and CD8 populations the same in our setting? We have previously demonstrated that in C57BL/6 mice, the memory lymphocyte population increases with age and express high levels of MHC class I on the cell surface.
Aim: To assess the CD4 and CD8 memory lymphocyte population according to HIV status in a population of patients with cervical cancer and controls.
Materials and Method: A total number of 61 females with cervical cancer and positive for the human papillomavirus (HPV) infection were examined at the Inkosi Albert Luthuli Central Hospital in Durban. Of these, eighteen patients (29.5%) tested positive for HIV. Peripheral blood mononuclear cells (PBMC) were stained with a quadruple antibody combination of antiCD3, anti CD45 RO (marker for memory cell population) anti CD45 RA (marker for naïve cell population) associated with either anti CD4 or anti CD8. Cells were analysed using Becton Dickinson flow cytometer (FACS calibur). Comparisons were performed by applying t-test (Instat 2 program, Graphpad).
Results: The overall percentage of memory lymphocytes was greater in older patients than in younger patients as previously described. In all patients combined, the average percentage of CD4 memory lymphocytes was 70±2%, statistically greater than the percentage of CD8 memory lymphocytes: 44±2% (p<0.0001). In the control population of women without cervical cancer, the percentage CD4 memory lymphocyte population was 63% while it was much lower: 34% in CD8 lymphocytes (p<0.0001). In the HIV negative group, the percentage of CD4 memory lymphocytes was 67±2% compare to 77±3% in the HIV positive group (p=0.01). A greater difference was observed in the CD8 memory lymphocytes: 40±3%
in HIV negative population versus 54±4% in HIV positive population p=0.0049).
Conclusion: These observations suggest that HIV infection either accelerates the maturation of CD4 and CD8 lymphocytes or preferentially destroys naïve lymphocytes.
Devi Sebastion with her poster which won first prize of R20 000.00
PRODUCTION OF A PROPHYLACTIC AND THERAPEUTIC VACCINE TO PROTECT AGAINST MULTIPLE HUMAN PAPILLOMAVIRUS TYPES
Marieta Burger, Gillian K. de Villiers, Inga I. Hitzeroth, Edward P. Rybicki Department of Molecular and Cell Biology, University of Cape Town
Background: Cervical cancer, the most common cancer affecting women in developing countries, is caused by persistent infection with high-risk types of human papillomavirus (HPV), most notably types 16 and 18, for which two vaccines have recently come on the market. However, there is still a need for a vaccine preventing infection by other types of HPV, especially in the developing world, where HIV and co-infection with multiple types of HPV is prevalent. Secondly, there is a need for a vaccine with therapeutic potential. Thirdly, current vaccines are unaffordable to the developing world.
Objective: The objective of this project is to produce chimeric L1 prophylactic and/or therapeutic vaccine candidates, containing L2 epitopes previously shown to elicit cross-neutralising antibodies, or E7 epitopes shown to be effective in immunotherapy, in insect cells using a baculovirus expression system.
Methods: Chimeric genes were designed by replacing segments of L1 with L2 and/or E7 peptides and synthesized by Geneart. Genes were cloned into insect cell expression vectors and their presence confirmed by PCR. All inserts were sequenced. Sf21 insect cells were transfected with recombinant bacmid or baculovirus shuttle vector DNA. Expression of proteins were confirmed by Western blotting. Proteins were purified by 24% Optiprep gradient ultracentrifugation. Future work includes structural confirmation by transmission electron microscopy, large-scale expression, large-scale purification, mouse immunogenicity studies, tumour regression studies and pseudovirion neutralization assays to assess cross-neutralising abilities of the chimeras.
Conclusion: All eight chimeric proteins were successfully expressed in insect cells. To date, one of the chimeras could be purified by 24% Optiprep gradient ultracentrifugation. This method can be further optimized in order to purify all eight chimeric proteins after large-scale expression for use in further studies. Using the most promising vaccine candidate, a variety of platforms will be assessed to determine the most cost-effective means of vaccine production.
LAMINA-ASSOCIATED POLYPEPTIDE 2 ALPHA (LAP2) EXPRESSION PATTERNS IN TRANSFORMED CELLS
Michelle Ward, Pauline van der Watt and Virna D. Leaner
LAP2a, a 75kDa isoform derived from the alternatively spliced LAP2 gene, contains a unique C-terminal tail important in binding both chromosomes and lamin A/C, present in the nuclear lamina, thus providing a structural framework in the nucleus as well as facilitating nuclear assembly and disassembly during the cell cycle. Gene expression studies identified elevated levels of LAP2a in cervical cancer patient material compared to normal. Altered expression of LAP2a may thus have significance in the development of certain cancers. The aim of this project is thus to confirm the up-regulation of LAP2a in cancer material and to determine the effect of this over expression on the biology of the cell. LAP2a expression was analysed in normal cervical tissue and cervical cancer patient material by Real-time RT-PCR analysis. Results confirmed the higher LAP2a expression in cervical cancer patient material compared to normal. Western blot analysis for LAP2a expression using proteins extracted from six cervical cell lines compared to that in a normal primary epithelial cell line, revealed a similar higher expression in the cancer cell lines. Interestingly, higher LAP2a expression appears to associate with cellular transformation as an increased expression was observed in HCX-E6/E7 transformed human cervical epithelial cells compared to the normal control cells (HCX). This pattern was also observed in normal and transformed fibroblasts, where the transformed fibroblast (SVWI38) showed significantly higher LAP2a levels compared to normal (WI38).
In summary, increased LAP2a expression associates with transformed and cancer cells. Its exact role in cancer progression and biology remains to be determined as well as its suitability as a molecular target in cancer treatment. To address this, studies using siRNA’s to inhibit its expression in cancer cells are currently underway.
DIFFERENTIAL EFFECTS OF SUTHERLANDIA FRUTESCENS SUBS. MICROPHYLLA ON CELL NUMBERS, MORPHOLOGY, GENE AND PROTEIN EXPRESSION IN A BREAST ADENOCARCINOMA AND A NORMAL BREAST EPITHELIAL CELL LINE
BA Stander, F Joubert, C Albrecht
Sutherlandia frutescens is a South African herbal remedy traditionally used for various ailments however little is known about the mechanisms of action of the constituents present in S. frutescens.
The in vitro influence of crude ethanolic S. frutescens extracts was examined in human breast adenocarcinoma (MCF-7) and non-tumorigenic breast epithelial (MCF-12A) cells. Dose-dependent studies were conducted on cell numbers and metabolic activity by means of spectrophotometry.
Morphological changes were determined with light-, fluorescent- and transmission electron microscopy (TEM). Cell cycle progression and apoptosis were analyzed using flow cytometry the differential effects of S. frutescens extracts on gene expression levels in both the MCF-7 and MCF-12A cells were conducted utilizing microarray analysis. mTOR kinase activity was measured with an ELISA assay.
S. frutescens reduced cell proliferation in both the non-tumorigenic MCF-12A and the tumorigenic MCF-7 cell line in a dose-dependent manner. The tumorigenic MCF-7 cells were more susceptible to S. frutescens treatment compared to the non-tumorigenic MCF-12A cells. Morphological characteristics of apoptosis and autophagy, including cytoplasmic shrinking, membrane blebbing and an increase in autophagic vacuoles were observed in both cell lines with the MCF-7 cells being more susceptible to autophagy and the MCF-12A cells less susceptible to autophagy and apoptotic cell death. TEM confirmed ultrastructural characteristics of autophagy in both cell lines. Flow cytometry revealed a G2/M arrest with no increase in apoptosis in MCF-7 cells and a G2/M arrest with an increase in apoptosis in MCF-12A cells treated with 1.5mg/ml S. frutescens extract. Microarray analyses revealed that the majority of S. frutescens-treated genes were down-regulated when compared to the vehicle-treated control and several genes involved in DNA replication and repair were differentially expressed in both cell lines. Abrogated mTOR kinase activity in both cell lines in response to S. frutescens extracts revealed by ELISA implicates attenuated mTOR kinase activity as a central mediator in inducing autophagy, suppressing gene expression and inhibiting ribosome biogenesis.
The current study contributes to the unraveling of the in vitro molecular mechanisms and signal transduction associated with 70% ethanolic S. frutescens extracts, providing a basis for further research on this multi-purpose medicinal plant in Southern Africa.
INFLUENCE OF 2-METHOXYESTRADIOL ON MORPHOLOGY, CELL CYCLE PROGRESSION AND CELL DEATH IN AN EPITHELIAL BREAST ADENOCARCINOMA AND A NORMAL BREAST EPITHELIAL
Vorster CJJ, BA Stander, AM Joubert
2-methoxy-17ß-estradiol (2ME2), an endogenous metabolite of 17ß-estradiol, is an anti-mitotic drug and tubulin poison, inhibiting growth and inducing apoptosis in a large variety of tumorigenic and non-tumorigenic cell lines in vitro and currently undergoing phase II clinical trials. These effects are observed in both estrogen receptor (ER) positive and ER negative cell types rendering 2ME2 as a potentially useful anti-tumour agent. However, several questions regarding 2ME2’s action mechanism and the basis for its differential effects on normal and tumorigenic cells remain unanswered.
The aim of this study was to evaluate the influence of 2ME2 on morphology, cell cycle progression and modes of cell death in the MCF-7 breast adenocarcinoma and non-tumorigenic MCF-12A breast epithelial cell lines. Fluorescence/PlasDIC-, transmission electron microscopy (TEM), flow cytometry, DNA gel electrophoresis and microarray gene expression studies were conducted after exposure of cells to 1×10-6M 2ME2 for 24h. Morphological analysis via fluorescence/PlasDIC microscopy and TEM revealed that both apoptosis and autophagy were induced in the MCF-7 cell line increased amounts of acidic vesicular organelles, membrane blebbing and hypercondensed chromatin in 2ME2-treated cells. This effect was not seen in the MCF-12A cell line. However, results did not indicate the occurrence of DNA laddering in either cell line after 24 h or 48 h of exposure. Flow cytometric analysis demonstrated an increased G2/M-phase and elevated cyclin B1 levels in 2ME2-treated MCF-7 cells.
Cell cycle progression and cyclin B1 levels were not significantly influenced in MFC12A cells. Bioinformatics analysis of microarray data indicated a statistically significant effect on the expression of 681 genes in the MCF-7 breast adenocarcinoma cell line including AKT1S, BAK1 and CALM2. These results denote that 2ME2 differentially affects morphology, cell cycle progression and cell death in the tumorigenic MCF-7 and non-tumorigenic MCF-12A cell lines.
Considering the potential benefits of the compound, further research into the molecular pathways involved in 2ME2’s action mechanism is warranted.
INFLUENCE OF NON-THERMAL 900 MHZ MOBILE PHONE RADIATION ON MORPHOLOGY, METABOLIC ACTIVITY AND DIFFERENTIAL GENE EXPRESSION IN A BREAST ADENOCARCINOMA AND NORMAL BREAST
S Marais, BA Stander, C Huyser, F le R Fourie, D Leszczynski, AM Joubert
Mobile phones and other hand-held type transceivers are widely used in the world and their utilization currently exceeds landline communication in Africa. This has raised concerns about the long-term health effects of their ongoing ever-increasing usage. In the present study, the in vitro effects of 1 hour exposure to 2W/kg non-thermal 900 MHz mobile phone radiation were assessed on morphology, metabolic activity and gene expression in breast adenocarcinoma (MCF-7) and normal breast epithelial (MCF-12A) cells. Light microscopy revealed no qualitative and quantitative differences in morphology of RF-exposed versus control MCF-12A and MCF-7 cells. Correspondingly, no significant differences in nuclear morphology were observed via fluorescence microscopy using propidium iodide and Hoechst 33342 staining. Statistically insignificant changes were observed in mitochondrial dehydrogenase enzyme activity in RF-exposed MCF-7 and MCF-12A cells respectively. Microarray analyses and bioinformatics analyses revealed 31 differentially expressed genes in the MCF-7 and 19 genes in the MCF-12A cell line. Genes involved in DNA repair in the MCF-7 cells include excision repair cross- complementing rodent repair deficiency complementation group 4 (ERCC4), DNA cross-link repair 1C DCLRE1C) and poly (ADP-ribose) polymerase family member 2 (PARP2) and chromatin assembly factor 1 subunit B (CHAF1B). Genes involved in cell differentiation, namely epithelial membrane protein2 (EMP2), germ cell-less homolog 1 (GMCL1) and BarH-like homeobox 1 (BARX1) were down regulated in the MCF-12A cells. Bioinformatics analyses are currently being performed on microarray techniques
Sumari Marais receives her poster award from Mrs Sue Janse van Rensburg, National Executive Director, CANSA
CHEMOPREVENTIVE PROPERTIES OF SOUTH AFRICAN HERBAL TEAS, ROOIBOS AND HONEYBUSH, UTILISING DIFFERENT CELL CULTURES AND SITE SPECIFIC RAT EXPERIMENTAL MODELS.
1Gamieldien, K., Joubert, E., Sissing, L., Marnewick J., Samodien S., de Kock, M and Gelderblom W.C.A.
1PROMEC Unit, MRC
Research on substances capable of modulating one or more phases in carcinogenesis, particularly dietary anti-carcinogenic compounds has recently gained popularity. Rooibos (Aspalathus linearis) and honeybush (Cyclopia intermedia) are herbal plants endemic to South Africa and their protective properties in various animal experimental models became evident recently.
The unfermented herbal teas showed a protective effect against carbon tetrachloride-induced hepatotoxicity, by normalising the clinical serum parameters associated with liver function, presumably by interfering with the drug metabolizing enzyme, cytochrome P450. In a two-stage mouse skin carcinogenesis model a lipophilic extract of both unfermented rooibos and honeybush, significantly decreased the size and incidence of the skin tumours whilst also delaying the onset of tumour development. Aqueous extracts of unfermented rooibos and honeybush, significantly reduced the number and size (multiplicity) of oesophageal papillomas induced by the site specific carcinogen methylbenzylnitrosamine. A similar effect was noticed in the rat liver model although the significant reduction of the incidence and size of preneoplastic foci was associated with an increased hepatotoxic effect during cancer promotion. In vitro studies, using the unfermented herbal teas and green tea (Camellia sinensis), showed differential effects against the cell viability of human liver (HepG2), skin (CRL-7762) and oesophageal (WHCO5) cancer cells. Unfermented rooibos and honeybush (C. intermedia) were the most effective in disrupting oxidative phosphorylation (ATP production) in the skin cancer cells, while rooibos also exhibited the highest activity in liver cancer cells. Rooibos and green tea displayed comparative effects in the oesophageal cancer cells, while honeybush exhibited the weakest response. Green tea exhibited the weakest activity in skin cancer cells, which was in contrast to its inhibitory effect against papilloma development in vivo.
Studies indicated that the anticancer properties rooibos and honeybush exhibit cell and organ specificity, presumably due to differences in their polyphenolic constituents.
Poster prize winners