Another post about honours

I haven't posted here for a while. But my honours is year is drawing to a close and today I presented my research to a room of my colleagues and several academic staff. I've always had an issue with nerves and today was no different. Everyone seemed confident and knowledgeable, and there I was, sweating beads and unable to formulate entire sentences. A few people nodded along, though, so I must have made some semblance of coherence.

My talk itself discussed the background to my research, based on cisplatin and several of its analogues, and presented what results I have so far (which I certainly won't be posting here). Cancer is such a huge and massive field that affects so many lives, and I feel fortunate to be partaking (however minutely) in the fight against it. Although many treatments for cancer exist, as we currently understand it, cancer is so overwhelmingly prevalent due to the fact that it is a naturally attractive state for individual cells to attain. Just as many humans aspire to immortality, the cells inside them may have already discovered what it takes to become immortal: becoming a tumour.

It's a bleak outlook to consider that when fighting cancer, we are fighting against natural selection itself.It's depressing to think that selection occurs at multiple levels, and that we are in constant battle against our environment and also our genes. Humans, let alone humanity, cannot achieve perfection, though perhaps it'd be nice to be proven wrong here. I don't expect I'd live to see that day.

Anyway, so far I haven't really talked about honours at all, oops. I suppose I did as well as I could have in my seminar, and since my supervisors have generally been impressed with my writing, it should give me some hope that my thesis would be more well received than my verbal presentation. I essentially completely failed in accomplishing half of my initial project aims, so that's a bummer there too. Hopefully if I stay on to do a PhD I'll be able to make something more meaningful happen out of that. Stupid DNA, get into this other DNA! I should get around to submitting my PhD application though. Sheeeeit I've been busy, all right?

Also I miss my sister.

:3

Identification of the DNA Adduct Sites of Cisplatin and its Analogues

Cancer claims the life of over 7 million people worldwide every year, a number that is set to rise over the next few decades. It is a disease that is diverse in its effects, targeting virtually every tissue in the body, yet are characterised by several common hallmarks (Figure 1). Hanahan and Weinberg in 2000 classified these as evasion of apoptosis, self-sufficiency in growth signals and insensitivity to anti-growth signals, sustained angiogenesis, limitless replication potential, and tissue invasion and metastasis. Metastasis refers to the secession of a malignant tumour from its central mass, entering the blood stream and re-establishing itself elsewhere.

Figure 1: Hanahan and Weinberg’s 6 Hallmarks of cancer

The current treatment regimes to combat cancer primarily consist of surgery, radiation therapy and chemotherapy, which will be the focus of this research. Cisplatin (Figure 2) is one of the most commonly used drugs in clinical cancer treatment, as it is highly effective against testicular, ovarian (the so-called “silent killer”), cervical, non small-cell lung, and head and neck cancers. Its mechanism of action involves the formation of adducts on DNA which hinder polymerase activity. However it possesses various toxic side effects, some of which are irreparable, and often meets with resistance by cells.

Figure 2: The chemical structure of cisplatin

Cisplatin is normally inserted into the body intravenously, where it will begin to diffuse into the cells. There is mounting evidence that certain proteins are involved in transporting it across the cell membrane. Once it enters the cell, an aquation reaction replaces one of the chlorine atoms, allowing this to form a bond with the N7 of a purine nucleotide, usually guanine. This is repeated to form a second bond, shown to occur most commonly on an adjacent guanine, but less commonly forms an ApG or GpNpG adduct, or form an interstrand adduct. The cisplatin adduct produces a kink (which various NMR studies show to vary between 40° to 60°) that hinders the progress of DNA polymerase along the template. Figure 3 represents this process. Unless repaired, this leads to apoptosis.

Figure 3: The binding mechanism of cisplatin on adjacent guanine nucleotides

Despite its widespread use, cisplatin has numerous toxic side-effects including neurotoxicity, gastrointestinal toxicity, nephrotoxicity (kidney damage), ototoxicity (hearing loss) and hematologic (blood) toxicity. Patients universally experience nausea and vomiting within hours of treatment, which can often be severe. Damage to renal function in particular is often irreparable, due to accumulation of cisplatin in the kidney.

Another cause for concern in the use of cisplatin is the development of resistance to the drug. This may be caused by a change in expression levels of genes which are thought to be responsible for cisplatin transport into the cells. There is some contention regarding whether or not cisplatin diffuses via active transport, and to what degree it occurs. It's possible that cisplatin does diffuse passively until a certain concentration, at which point further transport requires active protein assistance. Other mechanisms of resistance involve an increase in the proficiency of the repair mechanisms (primarily nucleotide excision repair) which are able to remove the DNA adducts. Increased expression of glutathione and metallothionein also contribute to cisplatin resistance due to competition for binding.

Other similarly related platinum compounds have also been developed in response to the toxicity and resistance problems. Carboplatin and oxaliplatin (Figure 4a) are currently used clinically worldwide, while nedaplatin sees more limited use in Japan. Carboplatin, while significantly reducing the toxic side-effects compared to cisplatin, has the same active form and is thus similarly unviable against cisplatin-resistant cells. Although cisplatin is able to bind onto DNA, it does so in a time-consuming manner since it has no natural affinity for nucleic acids. Thus analogues such as 9AmAcPtCl₂ (Figure 4b) are being studied, where the 9-aminoacridine moiety is able to intercalate with DNA.

a)

b)

Figure 4: The chemical structures for (a) carboplatin and oxaliplatin, clinically successful cisplatin analogues, as well as (b) 9AmAcPtCl₂, currently studied by the lab

My research will focus on comparing the adduct formation patterns of cisplatin, carboplatin and other analogues including 9AmAcPtCl₂ and related analogues, namely 7-methoxy-9AmAcPtCl₂, 7-fluoro-9AmA-cPtCl₂ and 9-ethanolamine-AcPtCl₂. Apparently my lab doesn’t currently have stocks of oxaliplatin but hopefully this will change soon. Due to the frequency of binding with GG dinucleotides, telomeric sequences (with their TTAGGG motif), CpG islands and other sequences of repeated guanines are expected to be preferentially targeted by cisplatin. Telomeres are of particular interest due to their expansion in tumour cells, while CpG islands are of interest due to their association with gene promoters. Specific promoters of genes of interest are proposed to be cloned into plasmids along with telomeric repeats and CpG islands. Several sequences have been previously analysed, including the transferrin receptor protein 1 (TFRC) and retinoblastoma protein (Rb1) promoter regions. These will be treated with cisplatin and analogues, then processed through a linear amplification assay using the fluorescent Rev2 primer (Figure 5) and sequenced to identify the location and frequency of DNA adducts formed by the platinum compounds. Fragment analysis will be performed at the Ramaciotti Centre.

Figure 5: The linear amplification method used to identify sites of cisplatin adduct formation

The results from the fragment analysis are presented as an electropherogram with peaks corresponding to the location of DNA adduct sites. The intensity of the peaks correspond to the abundance of adducts at that site. In this example set of data of T7.CpG.G10 DNA from Julie’s thesis (Figure 6), we can see that treatment with 3.0μM cisplatin produces mild peaks at the T7 region and higher peaks at the G10 and G5 regions, as expected with cisplatin’s preference for binding to tandem guanine repeats. The 3.0μM 9-aminoacridine analogue shows greater binding with the CpG region than with cisplatin. The right-most peak shows that a large amount of DNA remains unbound by the drugs at this concentration, as the linear amplification was able to continue to the end of the target sequence.

Figure 6: Example of an electropherogram obtained through fragment analysis of cisplatin-damaged DNA, in this case the T7.CpG.G10 plasmid construct.

A secondary aim will be to construct more plasmids to study, which will be started once I collect sufficient data from the cisplatin and analogue damage experiments on the presently available plasmids. Several lab techniques will be relevant to this aim which I would not have performed for the first set of experiments. DH5α E. coli cells will be grown on LB media and CaCl₂-treated to become competent for plasmid transfection. Following transfection, a midi-prep is performed to extract the plasmids from the cloned bacterial cells. The plasmid DNA is digested with PvuII to obtain the desired target sequence. This process is outlined in Figure 7.

Figure 7: Outline of the process involving construction of plasmids leading to cloning in DH5α cells. Plasmid extraction using a midiprep kit is carried out followed by digestion of the plasmid with PvuII to isolate the target sequence.

The genes of interest to be cloned are those which have been previously shown by microarray analysis to be differentially regulated following cisplatin treatment. These are BRCA2, MT1L and IL-1β, and have interesting properties besides being significantly regulated by cisplatin. Mutation in BRCA2 has been shown to be strongly associated with the development of breast and ovarian cancers, the latter being a clinically important target of cisplatin. As shown in Figure 8, women with BRCA2 mutations have a 23% overall lifetime risk of developing ovarian cancer.

Figure 8: Overall lifetime risk of ovarian cancer in BRCA1 and BRCA2-mutated women

The metallothionein gene product acts as a metal ion scavenger which can interact with cisplatin in the cell, such that increase in MT1L expression is associated with higher cisplatin resistance. There is some contention with this, however, and several studies agree that metallothionein expression levels do not affect cisplatin resistance to the same degree as glutathione. The interleukin-1β gene product is involved in the pathway to apoptosis, for example following cisplatin treatment. The interleukin-1β converting enzyme (caspase 1) has been previously shown to be induced by cisplatin.

So far I have produced an electropherogram of a linear amplification based on cisplatin-damaged T7.G10 DNA (Figure 9). Though similar to Julie’s results, it uses a slightly different DNA strand, essentially missing the CpG region between the telomeric repeats and the 10 tandem guanines. 30μM of cisplatin evidently produces so many adducts that virtually no strand of DNA was extended wholly.

Figure 9: Electropherogram of fragment analysis of T7.G10 plasmid treated with cisplatin, amplified with FAM-Rev2 primer

Cisplatin

So for my Honours year (starting in a month or so!) I'll be working on the cancer drug cisplatin (yay! Cancer treatment research!). Essentially this is a small platinum compound that binds to DNA, preventing its replication, thus inhibiting profileration of tumour cells. It has been employed quite successfully since the late 70's, after its anti-tumour properties were accidentally discovered (as all good scientific discoveries are) in a lab involving E. coli (oh E. coli, you sure do get around). Specifically it's one of the most commonly used drugs to treat testicular and ovarian cancer, and to a lesser extent, lung cancer and other cancers.

I'm not 100% sure where I'll be with the lab work, since my supervisors (Dr Anne Galea and A/Prof Vincent Murray; Prof Murray would have more on this on his university website if you care to look) have been working on this topic for years now so I'll jump along wherever they're up to in February. From what I understand it has to do with identifying the regions on the human genome where cisplatin binds to DNA. It's quite well established that cisplatin preferentially targets G-rich sequences (of ACGT fame), so the lab group intends to map out all such sequences in the genome via next-generation sequencing. These would be cloned into vectors and treated with cisplatin to measure the level of binding.

Additionally, quite a bit of work is being done on cisplatin analogues due to its toxic side-effects (nephrotoxicity, neurotoxicity, gastrointestinal toxicity, hematologic toxicity...). So similar compounds are being tested for their clinical activity in the hopes that they'll somehow be less toxic without sacrificing effectiveness. Mostly similar cis-structured platinum compounds with other branches to improve DNA-binding, etc. are being studied, and some appear to be moderately successful. It seems replacing the platinum core for a similar material like nickel, iridium or ruthenium doesn't work out the way researchers have hoped, though a group at USYD is still working on that, I think.

I've wanted to work on cancer treatment research for quite some years now (in high school I debated either this or marine biology), so I'm really excited that the opportunity is actually right in front of me. If all goes well, my plan is to do a PhD in the field and we'll see how it goes from there I suppose.

I wrote this instead of actually working on my literature review.