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.

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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