This research was carried out jointly by researchers from Hitachi Chemical Research Center, Inc., Irvine, Calif. 92617, USA and Epidemiology Division, Department of Medicine, University of California-Irvine, Irvine, Calif. 92697, USA.
A Sequence Listing is provided herewith.
1. Field of the Invention
The present disclosure relates to a method for determining cancer susceptibility by quantifying DNA damage-induced mRNA in whole blood.
2. Description of the Related Art
Cancer is caused by DNA mutation from exposure to DNA-damaging agents such as ionizing radiation, ultraviolet light, carcinogens, and free radicals, and by certain viral infections. Although cells successfully repair the majority of DNA damage, accumulation of uncured or miscured DNA damage at critical places within the genome may lead to the development of cancer. Thus, cancer susceptibility depends on the balance between DNA damage and corresponding cellular responses in a given individual. In fact, poor DNA damage response in ataxia telangiectasia (see Paterson, M. C. et al., Nature, 260, 444-47 (1976)) is known to frequently lead to the development of cancer. We first identified appropriate mRNA markers for DNA damage response and then applied the results to a clinical feasibility study.
Heparinized human whole blood from patients with invasive breast cancer, with (multiple primary) and without (single primary) a second primary cancer, and from unaffected controls was stimulated with 0.1-10 Gy of radiation and incubated at 37° C. for 2 hours. P21 and PUMA mRNA were then quantified. The results suggest that cancer susceptibility represented by the multiple primary cases was significantly related to over-reaction of p21 mRNA, and not PUMA.
Blood samples were collected from healthy adult donors (approved by the institutional review board (IRB) of APEX Research Institute, Tustin, Calif.). After treating the samples with 30 Gy of radiation (cesium-137), we first screened for expression of various mRNAs using a method we recently developed (see Mitsuhashi, M., Endo, K. & Shinagawa, A., Clin. Chem., 52, 634-42 (2006); Mitsuhashi, M., Clin. Chem., 53, 148-49 (2007)) with SYBR green real time PCR (see Morrison, T. B., Weis, J. J. & Wittwer, C. T., Biotechniques, 24, 954-62 (1998)) (
In these studies, heparinized human whole blood samples from five healthy individuals were stimulated with or without 30 Gy of radiation (cesium-137) and incubated at 37° C. for 4 hours. After incubation, triplicate 50 μL aliquots of whole blood were used to quantify various mRNAs by a method we recently developed (see Mitsuhashi, M., Endo, K. & Shinagawa, A., Clin. Chem., 52, 634-42 (2006); Mitsuhashi, M., Clin. Chem., 53, 148-49 (2007)) with SYBR green real time PCR (see Morrison, T. B., Weis, J. J. & Wittwer, C. T., Biotechniques, 24, 954-62 (1998)). Each gene was amplified individually. The cycle threshold (Ct)—the cycle of PCR that generates certain amounts of PCR products (fluorescence)—was determined using analytical software (SDS, Applied Biosystems). The melting curve was analyzed in each case to confirm that the PCR signals were derived from a single PCR product. The Ct values of drug-treated triplicate samples were subtracted from the mean Ct values of control samples to calculate ΔCt, and the fold increase was calculated as 2−ΔCt.
The results are shown in
Kinetic studies on p21 and PUMA mRNA were conducted using TaqMan real time PCR (see Holland, P. M. et al., Proc. Natl. Acad. Sci. U.S.A., 88, 7276-80 (1991)) with the values at time=0 as controls. The results are shown in the inset of
We hypothesized that cancer susceptibility might be linked to hypo-functions of p21 and/or PUMA, based on our knowledge of ataxia telangiectasia (see Paterson, M. C. et al, Nature, 260, 444-47 (1976)). To test this hypothesis, we undertook studies to evaluate the blood from control and cancer patients for inducibility of p21 and PUMA mRNA after radiation exposure. After obtaining approval for the study protocol from the IRB of the University of California-Irvine (UCI), we identified 38 cases in the local cancer registry where the patient had both invasive breast cancer and a second primary cancer (multiple primary cases (MP)). After initial contact, we recruited 21 women to participate in the study. We then selected a second cancer group of single primary cases (SP) (n=21) and unaffected control cases (UC) (n=20) with similar age and ethnicity distributions. Table 2 provides demographic and tumor characteristics of the recruited participants, and Table 3 shows their white blood cell (WBC) counts:
We dispatched clinical nurses to the participants' homes to complete questionnaires, and blood was drawn in two tubes from each participant, one for a complete blood count (see Table 2) and the other for mRNA analysis. Blood samples were immediately transferred to the laboratory at 4° C. The blood was treated the same day with radiation (2 hours at 37° C.), and the samples were then frozen at −80° C.
Specifically, the heparinized human whole blood samples from invasive breast cancer with or without a second primary cancer, or unaffected control (◯, n=20,
The results are reported in
As shown in
The population density was shifted upward for radiation-induced p21 mRNA in MP cases for all doses of radiation as compared to the other two groups. For example, three (14%) and four (20%) individuals in SP and UC cases respectively showed a greater than two-fold p21 mRNA induction at 0.1 Gy, whereas the percentage was significantly higher in MP cases (57%)—this difference was statistically significant (p=0.004 (MP v. SP), p=0.01 (MP v. UC) by χ2 test, respectively) (
As discussed above, we had initially hypothesized that cancer susceptibility might be linked to hypo-functions of p21 and/or PUMA, based on our knowledge of ataxia telangiectasia (see Paterson, M. C. et al., Nature, 260, 444-47 (1976)). Surprisingly, the results suggested that cancer susceptibility is related to the over-reaction of p21 mRNA only, and not PUMA (see
Cancer susceptibility is currently analyzed extensively from a genomics perspective in order to identify specific single nucleotide polymorphisms (SNPs) (see Karlan, B. Y., Berchuck, A. & Mutch, D., Obstet. Gynecol., 110, 155-67 (2007); Oldenburg, R. A. et al., Crit. Rev. Oncol. Hematol., 63, 125-49 (2007); Naccarati, A. et al., Mutat. Res., 635, 118-45 (2007)). However, we still do not know whether yet-to-be discovered second or third SNPs will compensate for or aggregate the effects of a given SNP. By contrast, we quantified the levels of normally existing mRNA without considering SNPs in p21 and PUMA. The hyper-function of p21 mRNA that we found may result from an SNP in p53 or other related genes. Alternatively, it may be related to the strength of each participant's antioxidant levels, which protects against DNA damage. We have thus generated a unique model for cancer susceptibility research as a screening tool for various downstream molecular assays.
All references cited herein are expressly incorporated by reference.
This study was funded in part by a grant from the Hereditary Breast Cancer Research Project (NIH # CA-58860-12). The government may have certain rights in the invention.
Number | Date | Country | |
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61032020 | Feb 2008 | US |