ADP-ribose polymer - a novel and general biomarker of human cancers of head & neck, breast, and cervix

Poly-ADP-ribosylation is an enzyme catalyzed reversible post-translational modification of cellular proteins [reviewed in [1‐5]]. During this process, ADP-ribose units are transferred onto or stripped off the acceptor or target proteins. Accordingly, the target protein becomes poly-ADP-ribosylated or deribosylated, respectively. The biosynthesis of the homopolymer of ADP-ribose or poly-ADP-ribose adduct(s) is primarily catalysed by the enzyme, poly-ADP-ribose polymerase (PARP), which is ubiquitously present in the nuclei of metabolically active cells [1, 5, 6]. The enzyme progressively and sequentially catalyses transfer of ADP-ribose units derived from the endogenous nicotinamide adenine dinucleotide (NAD⁺) donors onto acceptor or target proteins creating homopolymeric adducts in a variety of linear and branched architectures. Nuclear proteins, particularly histones, are the primary targets of ribosylation [3‐5]. Poly-ADP-ribose glycohydrolase (PARG) is the main enzyme involved in the breakdown of adducts from the protein [1, 3, 4]. The build up of the adduct on or its breakdown from the chromosomal proteins reversibly alters chromatin superstructure and, thereby, significantly influences the pattern of gene expression [1‐5]. Consequently, status of ribosylation of chromosomal proteins is intricately involved in carcinogenesis [4, 5]. Previous detailed investigations done in murine model have shown that the metabolic level of the ADP-ribose adduct on total cellular proteins is significantly reduced during different stages of carcinogenesis [7‐13]. These systematic studies have revealed the existence of an inverse relationship between carcinogenesis and the total ADP-ribose polymers or poly-ADP-ribose adduct level measured either in cancerous tissue or in peripheral blood lymphocytes (PBL). Therefore, we hypothesize that a decreased quantum of the total adduct of poly-ADP-ribose on human PBL proteins would also correlate with human cancer. In order to test the hypothesis, we have detected and quantified the total concentration of ADP-ribose polymer adducts of PBL proteins in healthy individuals (controls) and cancer patients (cases). PBL was chosen since PBL proteins have been shown to mirror the status of ribosylation of cellular proteins of liver, spleen, ascites cells and other tissues [10]. In addition, a blood based assay of ADP-ribose polymer adducts is the most convenient procedure to perform on humans since it is the most non-invasive medical intervention. An assay using other tissues or biopsies pose practical problems as obtaining the tissue usually requires surgical intervention. Furthermore, the immune system of the body resides in the blood. The PBL are the first responders to foreign invasions across the body. They also communicate with cells as well as cellular matrices of all tissues/organs of the body [14]. This circulating tissue, therefore, qualifies to be the sentinel of the whole body [15]. We have chosen the PBL for this reason as our aim was to identify and establish a general biomarker of cancer, which can be conveniently monitored.

This hypothesis has been tested in total cellular proteins of the PBL of normal human donors (controls) and in patients (cases) with advanced head & neck (H & N), breast or cervical cancers using a novel immunoprobe assay that quantitatively measures the total cellular poly-ADP-ribose polymer [5, 7, 8].

Blood samples were collected from new cancer patients (cases), who had not received any treatment so far, as well as from healthy volunteers (controls) in accordance with the approvals of Institutional Ethics Committees of NEHU and BBCI. The cases comprised male as well as female patients with advanced stages of cancers at 14 different sites of head & neck region (grouped as Ca H & N), breast and cervix (age range 25-80 years). The controls consisted of healthy, male and female volunteers with no known history of cancer (age range 25-80 years). Analysis of age distribution of controls and cases was done using unpaired t-test with Welch correction and χ² test, which showed that statistically older males (P ≤ 0.0001), but not females (P = 0.4250), made the case group (Figure 1 - inset Table).

Overall, the cancer patients (cases) were statistically older (47.70 years) than the control counterparts (39.90 years) (Figure 1 - inset Table). The PBL protein samples (500 ng per slot) were prepared from blood samples of controls (n = 68) and cases (total n = 112). The cases comprised patients with cancers of 14 different sites grouped as Ca H & N (total n = 66), Ca breast (n = 22) or Ca cervix (n = 24). Samples were subjected to quantitative detection of poly-ADP-ribose adducts using the novel slot blot immunoprobe assay followed by densitometric analysis as described [7, 8]. Figure 1 - a shows a typical slot blot stained for total proteins using India ink (left panel) and its immunoprobed replicum (right panel) for five replicate samples from a control subject (A) and a cancer patient (B). The concentration corrected mean net intensity of each control sample has been plotted as a dot to show the distribution of measured poly-ADP-ribose adduct levels in relation to sample number (Figure 1 - b), age of patient (Figure 1 - c) and gender of patient (Figure 1 - d) highlight the range of variation in measured control values in arbitrary units (AU) (mean: 14573 ± 1452 AU; range: 11950 - 17540 AU).

Compared to the control group, the total poly-ADP-ribose adduct of PBL proteins of H & N cancer patients showed a statistically significant (P < 0.0001) reduction in cancers of all 14 sites (Figure 2 - a, inset Table).

The overall reduction was approximately 45% with a mean of net intensity of 8060 ± 707.10 AU (range of 6324 - 10318 AU). Cancers of all sites in this group individually (Table 1) as well as collectively (Table 2) showed significant reduction as revealed by Student's t-test and ANOVA, respectively. Similarly, there were significant reductions in the total poly-ADP-ribose adduct of PBL proteins in cancers of breast (Figure 2 - b, inset Table) and cervix (Figure 2 - c, inset Table) in comparison to all controls (males + female controls combined) as well as only female controls. The extent of reduction in the poly-ADP-ribose adduct among the cases was 47% and 43% for breast and cervical cancers, respectively, which are statistically highly significant (P < 0.0001, unpaired t-test with Welch's correction) as shown in the inset table (Figure 2). These results are in line with previous studies done in murine model, which showed negative and statistically significant correlation between total cellular poly-ADP-ribose adduct and carcinogenesis [8‐13].

As this report deals with elucidation of a general biomarker of cancer, the mechanistic aspects of lowering of the level of poly-ADP-ribose adduct of cellular proteins and its likely implications on the biological functions of chromatin, including gene expression, is not being discussed here. These aspects have been reviewed in details recently [5] as well as earlier [4]. It might be prudent to add that in the our considered opinion, the PBL, being the first responders of the body to sense metabolic changes and mount appropriate biological response(s), are the most appropriate cells to look for the alterations in the cellular concentration of the ADP-ribose polymer during carcinogenesis.

The subjects under investigations are likely to be exposed to different and variable etiological factors. Etiological factors could influence both measured poly-ADP-ribose adduct level as well as the course of carcinogenesis. Therefore, we also analysed the influence of three most important etiological factors relevant to the study on poly-ADP-ribose adduct and human cancer. The factors are habits of (a) wet variety of betel nut (BN) chewing - a social etiquette that is strongly associated with cancers, especially to cancer of H & N [reviewed in [16, 17]], (b) alcohol consumption [reviewed in [18]] and (c) tobacco usage [reviewed in [19, 20]].

These three factors did not show any statistically significant influence on the level of poly-ADP-ribose adduct in control subjects as revealed by Spearman rank correlation and Mann-Whitney rank sum test (Table 3). However, while BN chewing did not significantly influence the total poly-ADP-ribose adduct of PBL proteins in cancer patients (Figure 3 - c), alcohol consumption (Figure 3 - a) and tobacco usage (Figure 3 - b) were found to lower the level of total poly-ADP-ribose adduct of PBL proteins in cancer patients significantly as compared to the patients without these habits (Table 4).

In order to further compare the total poly-ADP-ribose adduct of PBL proteins in controls and cases, we matched subjects for age and gender. We obtained 28 controls (11 females and 17 males) and 56 cases (33 females and 23 males) in the age range of 24-65 years (Figure 3 - d, e and inset Table). Again a statistically highly significant (P < 0.0001) reduction (~ 47% in males and ~ 44% in females) in total poly-ADP-ribose adduct of PBL proteins was observed in cancer patients when compared with their age and gender adjusted controls.

In conclusion, our findings reveal statistically highly significant negative correlation between total poly-ADP-ribose adduct of PBL proteins in advance human cancers of H & N (14 different types), breast and cervix in comparison to healthy controls using a novel immunoprobe assay of poly-ADP-ribose adduct. Based on the preliminary data, we conclude that poly-ADP-ribose adduct of PBL proteins is a useful and general biomarker of cancers in humans. Potentially it can be used as a convenient tool for detection of cancer using the novel immunoprobe assay. The ease with which the assay could be performed along with the results presented in this report support our earlier contention [8, 10‐13] that the total poly-ADP-ribose adduct of PBL proteins can potentially be used as a tool for mass screening of cancer. We are aware of the fact that further studies on patients in early stages of different cancers and patients undergoing chemo- or radiotherapy, etc. would strengthen the reported findings.