Sample Two

Harvard


Topic: toxicity of cadmium chloride in leukemia cells and the cadmium induced the HERV-W in leukaemia cells

Order type: Research Paper Deadline: May 23 12:32 Pages: 12 , Double spaced Sources: 22

Subject: Biology Academic Level: PhD Style: Harvard

Toxicity of Cadmium Chloride in Leukaemia Cells and the Cadmium induced HERV-W in Leukaemia Cells (Name) (Institution)

Toxicity of Cadmium Chloride in Leukaemia Cells and the Cadmium induced HERV-W in Leukaemia Cells

Section A: Results Cadmium chloride is a compound made up of cadmium and two chloride ions. Its chemical formula is CdCl2. It is hygroscopic, has an average mass of 183.317001 Daltons and a monoisotopic mass of 183.841064 Daltons (www.chemspider.com). Other chemical properties include the ability to dissolve in water and alcohol, although to a slight degree in the latter. Initial studies that have shown that cadmium chloride has effects on cell viability, particularly leukaemia cells prompted the need for this study. The compound has widely been known to be an environmental pollutant. However, recently, it has emerged to be one of the few compounds that effectively inhibit the viability of cancerous cells (Banfalvi, 2011). For instance, studies that have been conducted reveal that cadmium chloride has growth inhibitory effects on MDA-MB468 cancer cells of the human breast (Panjehpour et al, 2011).

In this study, six cell lines were selected to represent a model of leukaemia cells for CdCl2 toxicity. The six cell lines were Jurkat, K562, CCRF-CEM, FA-AML, OC-AML and MOLT4. The study produced somewhat mixed results although a number of the cell lines produced positive results. It (the study) revealed that CdCl2 significantly reduced the viability of Jurkat, MOLT4, K562 and FA-AML. However, the CCRF-CEM and OC-AML cell lines showed resistance towards CdCl2.

The study involved a 24, 48 and 72 hour period within which the different cell lines were exposed to 0, 10, 20 30 and 40 micromole concentrations of CdCl2. This is shown in figures 1 and 2. From the results, there was a significant drop in cell viability after treatment with 30 and 40 micromoles of CdCl2. The treatment was done for 24, 48 and 72 hours respectively (Fig 1A, B, and C). The results showed that Jurkat and K562 cell lines were the most sensitive and vulnerable at a higher concentration of CdCl2. The results indicate that their viability was reduced by up to 50 per cent (fig 1 and 2). However, a marked cytotoxic effect was observed when K562 cells were treated with 40 micromoles of CdCl2.the results showed decreasing cell viability with 69% after 24 hours, 37% viability after 48 hours and 24% viability after 72 hours (Fig 1B). However, for FA-AML and MOLT4 cells, the sensitivity was lower. Even then, FA-AML cells were more sensitive than the MOLT4 cells (Fig1C, 2A). In FA-AML cell lines, a clear inhibition of cell proliferation after exposure to different concentrations of CdCl2 was observed after 48 and 72 hours.

From the results, the longer the incubation period the higher the toxic effect inflicted on the four cell lines (Jurkat, K562, MOLT4 and FA-AML leukaemia cells). Nevertheless, all the cells showed different levels of sensitivity to the toxic effects of CdCl2 indicating the difference in cell types and genetic factors (from the results).

Unlike the first four cell lines, CdCl2 did not alter cell survival in CCRF-CEM and OC-AML cells notwithstanding its concentration. Even the 40 micromole CdCl2 had no effect. The CCRF- CEM and OC-AML cells had no statistically significant cytotoxic effect shown as compared to the control. However, there were some surprising findings, most notably the difference in resistance to Cd in CCRF-CEM cells. This is shown in figure 2 C. From the results, these cells were more resistant to 20 and 40 microlitre CdCl2 for 24, 48 and 72 hours as compared to lower effective dose.

(A) (B) (C)

Figure (1). CdCl2 toxicity to cell lines. The graphs show effects of cadmium chloride with different concentration and incubation period (24, 48, 72hr) on viability of Jurkat (A), K562 (B) and FA-AML (C) cells. The results are mean ± SD of three independent experiments, with*p < 0.05 and **p < 0.01 compared to the control.

(A) (B) (C)

Figure (2): Proliferation curves of MOLT4 (A), OC-AML (B) and CCRF-CEM (C) cells treated with different concentration of Cadmium chloride for 24, 48 and 72hr incubation. Each values were expressed as the mean ± SD of three independent experiments, with*p < 0.05 and **p < 0.01 compared to the control 3.2 Induction of HERV-W Gene Expression by CdCl2

There are a potential role of endogenous retroviruses in the pathogenesis of leukaemia has been discovered earlier (Zur Hausen, 2006). Expression analyses of HERV-Wenv gene in six leukaemia cell lines were performed by RT-PCR. The aim of this experiment was to investigate the effects of 24 hour CdCl2 treatment on Expression of HERV-W env in leukemia cell lines. (Fig.3).As shown in Fig (3), RT-PCR analysis demonstrated that HERV-W env transcript levels varied across the cell lines which might be related to different types of leukemia. Jurkat, K562, CCRF-CEM and OC-AML cells had a constitutive expression of HERV-W env transcript even with treated with CdCl2. Jurkat, K562 and OC-AML had approximately similar HERV-W env transcript levels. But, the CCRF-CEM cells transcribed only half the level of HERV-W env transcript.

Figure (3): RT-PCR analysis of mRNA for the expression of GAPDH and HERV-Wenv gene from various leukaemia cell lines (Jurkat (JK), K562, FA-AML, CCRF, OC-AML and MOLT4) after treatment with 20 µM CdCl2 for 24 hours. Distilled water was used as negative RT- PCR control and RT PCR negative control (-CDNA) specific to each cell. * Constitutive expression of HERV-Wenv in these cell lines.

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Figure (4): FA-AML and MOLT4 cells were incubated with 5, 10 and 20µM CdCl2 for24hrs, and total RNA was isolated and subjected to RT-PCR analysis using GAPDH and HERV-Wenv primers (table 1). Distilled water was used as negative RT- PCR control and RT PCR negative control (-CDNA) specific to each cell. Results shown are representative of three independent experiments.

3.3 Effect of CdCl2 on the Expression of HERV-W env (Synctin) proteins This experiment was designed to test whether HERV-W protein expression was consistent with the previously described RT-PCR results. It was important to investigate protein expression in all HERV-W mRNA positive cells to determine if the protein was also induced. Cell lysate prepared from FA-AML and MOLT4 cells treated with and without CdCl2 and were analysed with 10% SDS-PAGE gel and western blotting analysis of GAPDH and HERV-W in FA-AML and MOLT4 cells after CdCl2 treatment. Western blot analysis was carried out using primary rabbit anti HERV-W env and anti-GAPDH was used as a loading control. Figure 7 shows several interesting features: (1) GAPDH was not affected by CdCl2 in FA-AML and MOLT4 cells. (2) Different concentration of CdCl2 (5 ,10 and 20µM) obviously induces the level of HERV-W protein significantly in the FA-AML and MOLT4 cells as compared with untreated cell (control) (Fig 7A). (3) It has shown a slightly expression of HHERV-W protein in FA-AML and MOLT4 control (untreated). (4) Quantified by densitometry. Western blots were analysed densitometrically, normalized to GAPDH, as shown the HERV-W env protein has significantly increased after exposure to 5, 10 and 20µM of CdCl2 gradually for 24hrs in both FA-AML and MOLT4 cells (Fig 7B).

B Discussions Cadmium, a toxic metal acts by various mechanisms to induce cell damage. Some of these mechanisms are still not well understood and further investigations are still ongoing (Yamada et al, 2009). However, its toxicity appears to be influenced by the expression of the 78-kiloDalton glucose regulated protein Grp78. This is a member of the heat shock protein 70 (HSP70). This family of chaperones is found in the cytoplasm. The silencing of the Grp78 expression appears to be the mechanism of action by which CdCl2 induces cell damage in renal cells. This mechanism is likely to be the same witnessed in leuakaemic cells. Variation in the Grp78 due to genetic factors or cell origin could play role in some of the cells being sensitive to CdCl2 toxicity while others are resistant (including CCRF-CEM and OC-AML). CdCl2 also appears to induce cell damage via apoptosis and by inducing m RNA levels (Yamada et al, 2009).

In a study conducted to evaluate the effects of CdCl2 on gene transcriptions, it was found that it causes up regulation of genes at a higher concentration of say 20 and 40 microlitres. On the other hand, it causes down regulation of the same genes more effectively at a lower concentration of 10 microlitres (Tsangaris et al, 2004). In a related study involving the LLC-PK1 cells, 10 micromoles of CdCl2 increased the levels of Grp78 significantly after 6 hours. The same test conducted with 20 micromoles revealed that the levels of Grp78 increased depending on the dose (Tsangaris et al, 2004). The ability of the lower concentration to down regulate the expression of genes could be an important factor in inhibiting cell viability in CCRF-CEM cell lines. The down regulation of genes could also cause interruptions in a number of subcellular compartments thereby contributing to the overall inhibition of the cell viability. Further, the interruptions caused to these compartments may alter cell functions (Yamada et al, 2009).

CdCl2 is not only toxic to leukaemic cells but also to other mammalian cells as well (Schwerdtle t al, 2010). Cadmium is better absorbed in the lungs than in the gastrointestinal tract. For this reason, inhalation of compounds that contain cadmium significantly increased the risk of lung cancer. There are numerous postulations as to how it increases this risk. However, one common postulation is that it produces reactive oxygen species and together with the inhibition of DNA repair, offsets a series of reactions that lead to the production of cancerous cells (Greim, 2005). In addition, it is distributed in the body while bound to red blood cells. This is part of the reason why it affects many organs and systems in the mammalian body. The two organs in which it accumulates are the kidneys and the liver (Banfalvi, 2011). Studies reveal that cadmium plays an active role in tubular epithelium damage and its regeneration. Although the mechanism by which this occurs has not been comprehensively clarified, it has been postulated that CdCl2 acts by exerting stress on the endoplasmic reticulum (Joseph, 2009). This is the organelle responsible for protein synthesis. Inhalation and/or ingestion of cadmium by a pregnant woman may spell gross defects in the unborn baby. Although studies show that these defects may not exactly be teratogenic, it is indicated that the skeletal system may grossly be affected. This effect is mainly the result of altered osteoblastic cell functions and mutations induced by the toxic effects of cadmium. Moreover, the neonates may have lower birth weights. In the reproductive system, studies reveal that adult males exposed to cadmium have a reduced sperm count and motility. In addition, they present with atrophied seminiferous tubules (Joseph, 2009).

One of the reasons for the fatality of cadmium exposure is that once inhaled or swallowed, it undergoes very little metabolism if at all. As a result, the much that is not excreted ends up accumulating in the body and exerting its toxic effects (Joeph, 2009).

Expression the HERV-W in Leukaemia Cell

The Human Endogenous Retrovirus W (HERV-W) is a newly described member of the family of the human endogenous viruses. HERVs form approximately 8 per cent of the human genome (Bannert & Kurth, 2004). They are important in analyses aimed at detecting the presence of viruses or other medical conditions that involve attacking and undermining the functionality of human cells. This is because HERVs together with other replication defective viral particles can be detected in tissues in normal or pathological conditions (Griffiths, 2001).

It has been discovered that endogenous retroviruses play a role in the pathogenesis if leukaemia. The HERV-W has gag, pol and env structural proteins. The gag and pol genes are selectively expressed and are only found in specific cells. However, the env gene is common and is found in most cells, including normal and cancerous cells (Griffiths, 2001).

The envelope glycoprotein (env) appears to perform a number of positive roles as long as its expression is control and regulated adequately (Griffiths, 2001). The difference in expression of the transcript m RNA could be attributed to different types of leukaemia.

2 HERVs are frequently expressed in normal tissues. Here, they play a number of positive roles including induction of resistance to invasion of the exogenous retrovirus and enhancing local immunosuppression (Griffiths, 2001). The former occurs through receptor interference. In addition, they allow the formation of connections (syncytia) between adjacent cells, thereby enhancing cell-cell fusion a factor that is important in cell differentiation (Griffiths, 2001). Thus, abnormal expression of HERVs deprives the host cells of all the above attributes. More so, HERV-W env performs the above functions among a host of others that overall aid in limiting viability and spread of cancerous cells. As such, when HERV-W env is abnormally expressed, the likely result is increased viability of cancerous cells due to a weakened cell defence mechanism and an even weaker viral response system. Reverse transcription polymerase chain reaction (RT-PCR) is a qualtitative technique used in the detection of RNA expression levels. It detects RNA (gene) expression by creating complementary DNA transcripts (c DNA). It also acts as an amplifier, significantly multiplying DNA sequences. On the other hand the Western blot technique is a method of analysis that involves the removal of a biological sample from a gel and placing on a membrane. From this membrane, detection of the protein can be made. The technique is based on the fact that antigen-antibody interactions are specific and this information is used to separate proteins from a mixture. The aim of conducting both the RT-PCR and the Western blot analysis was to investigate the induction of proteins together with the HERV-W env.

A B

As earlier outlined, HERVs have a number of important roles in the body. One of these is playing a vital role in the differentiation and cell fusion in the trophoblast, a factor that is important in the formation of the placenta (Griffiths, 2001). However, HERVs have also been associated with a number of pathological conditions most notably cancer and neurogenerative diseases (Griffiths, 2001). Cancers and neurodegenerative disease are more often than not the consequential results of reduced expression of HERV proteins. In such instances, the protective mechanisms that HERVs bestow on the body cells and that have already been mentioned above are lacking. Thus, the ability of cancerous cells to maintain viability and to multiply is favoured (Banfalvi, 2011). These and other conditions are more often than not the consequence of reduced expression of these proteins. However, there are circumstances when over expression of these proteins in itself leads to a pathological condition. An example is in multiple sclerosis in which a high level of HERV-W env expression is found in patients who are in the active phase. Gag and env proteins are usually expressed in normal neuronal cells. In a study conducted to determine the impact of HERV expression in multiple sclerosis, it was found that there was physiological expression of gag proteins in the neuronal cells in a normal brain. On the other hand, in patients suffering from multiple sclerosis, there was an acute accumulation of gag proteins in the axons in demyelinated white matter. Increased HERV-W gag expression was also detected in endothelial cells of lesions caused by multiple sclerosis in active or acute phases (Dixon & Kunkel, 1982).

HERVs are present in both normal and cancerous cells (Griffiths, 2001). Some factors and viruses have been shown to cause activation of the HERV-W in human cells. Such factors include certain exogenous viruses and toxoplasma (Kurth &Bannert, 2010). The presence of certain amino acids such as lysine for HERV-K and histidine for HERV-H may also culminate to activation (Yamada et al, 2005). Moreover, cancer and other inflammatory diseases such as systemic lupus erythematosus and chronic inflammatory disease may also act as activating factors.

Cadmium is a heavy metal that causes acute toxic effects following exposure. Exposure may arise from ingestion or inhalation. The World Health Organization states 0.5 mg/m-3 as the highest amount that can cause respiratory effects if the individual is exposed for a period of 8 hours. Exposure to 1-5 mg/m-3 of cadmium is identified as ‘immediately extremely dangerous’. A dose higher than this may result to various effects on the body including oligouria, pulmonary oedema, metabolic acidosis, altered metabolism of calcium and severe fluid loss among others(Schwerdtle et al, 2010) . However, the most important effect of cadmium in mammalian body cells is arguably the ability to cause cancer. It has often been linked to numerous cases of lung cancer (Schwerdtle et al, 2010). In HeLa, Chang and HCT15 cell lines, cadmium induces its carcinogenic by affecting translation initiation factor 4E (eIF4E). The resultant effect is cytotoxicity and consequently cell death. A reduction in eIF4E protein levels causes cell death (Yamada et al, 2005). Cadmium compounds also induce growth of tumours in prostate, lung and testes (Schwerdtle et al, 2010). More directly related to cancer, they cause chromosomal aberrations, morphological transformations and mutations of genes. They also induce damage to chromosomes and cause breakage of DNA strands (Yamada et al, 2005). These factors together culminate to development of cancerous cells. Cancer occurs due to gene mutations that give normal cells the ability to grow and multiply exponentially. In normal cells, growth and development of cells is closely regulated and is followed by apoptosis shortly after.

Due to its carcinogenic effects and other toxic effects on the body, exposure to cadmium causes various pathological states (Banfalvi, 2011). Lower doses of cadmium may have mild systemic effects such as vomiting and nausea. However, larger doses are more fatal and cause problems in the cardiovascular system, the renal system and the nervous system among others (Greim et al, 2005). In the nervous system, exposure may cause motor function disturbances, peripheral neuropathy and amyotrophic lateral sclerosis (Greim et al, 2005). Studies reveal that the effects of cadmium toxicity may not be apparent until long afterwards. For instance, a worker exposed to toxic levels of cadmium for about an hour may present with impaired lung function four years down the line (Hartwig, 2010).

Leukaemia, which is cancer affecting the white blood cells is rare yet fatal. It is caused by various genetic and environmental factors that predispose individuals and make them susceptible (Bain, 2010). One of these is cadmium. Through the mechanisms explained above, it offsets a series of reactions beginning with effecting gene and chromosomal mutations. This eventually culminates to leukaemia (Zenz et al, 2005).

Other than the cancer of the lungs and leukaemia, cadmium has also been associated with cancers of the prostate, kidney, liver and haempoietic system (Hartwig, 2010). Most of the cadmium in the body (50-70%) accumulates in the liver. Since there is little metabolism of this compound, accumulation in these organs only means that there is enough time and chemical activity to instigate carcinogenic effects on these organs (Hartwig, 2010).

Reduced expression or over expression of HERVs may have a negative consequence on the normal functioning of body cells (Griffiths, 2001). However, when correctly expressed, these proteins are beneficial to the body. Increased expression particularly causes pathological conditions such as multiple sclerosis. From the results of the tests done above, it was found that cadmium chloride (and probably other cadmium-containing compounds) causes marked increase in HERV-W levels. Further, the relationship between the two was dose related. This means that an increase in cadmium chloride in the body cells resulted to marked increase in the levels and expression of HERV-W levelsTherefore, part of the explanation for the rise of leukaemic cells due to exposure to cadmium could be the increased expression of HERV-W in different cell lines, a factor that strips the body of their protective effects. Immunosuppression may also result from this paving way for development of cancerous leucocytes. Studies reveal that there is a high incidence of neoplasm of the immune system itself in relation to cancer (Penn, 1986).

Expression of HERV W env was not associated with acute toxicity in MOLT4 cells treated with cadmium. According to results from the first part of the experiment, other cell lines including K562, Jurkat, CCRF-CEC, OC-AML and FA-AML were comparatively sensitive to cadmium as compared to MOLT4. There was little inhibition of proliferation of these cells compared to other cell lines. Inhibition of viability and growth of these cell lines results to cytotoxicity and death of cells. However, since the MOLT4 cells are resistant, they remain viable and therefore remain physiologically functional. This reduces the chances of acute toxicity. Another line of thought could be in the expression of HERV-W env proteins in MOLT4 cells.

DNA sequencing confirmed the HERV-W expression in leukaemia. The results from the study showed that 82% of DNA sequencing was similar to HERV-W gene in leukaemia cells. The identity was illustrated by blast analysis using Gene Bank database. These results point to the fact that MOLT4 is less sensitive to cadmium chloride and possibly other cadmium compounds. The explanation for this relationship is not clear. However, some researchers have claimed that it could be a difference in genetic make-up (Taruscio et al, 2002).

Conclusion

Cadmium chloride it toxic to mammalian cells as has been noted in this study. It has varying effects on different cell lines and this could be due to variation in the expression of the HERV- W env protein. It has been postulated that this compound acts by various mechanisms to cause toxicity and carcinogenicity in these cells including interference with the functionality of the endoplasmic reticulum. Even then, some of the mechanisms by which it acts are not clearly understood and research is still ongoing. Future research should focus on the postulated mechanisms by which the compound acts and other mechanisms not yet suggested. This may go a long way in helping in the discovery of drugs that can be used to manage leukaemia and other cancers caused by cadmium chloride and related compounds.

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