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Expression of ras (p21) protein in plasma from exposed workers and from patients with lung disease
Int. J. Hyg. Environ. Health 204, 55 ± 60 (2001) Urban & Fischer Verlag http://www.urbanfischer.de/journals/intjhyg
International Journal of Hygiene and Environmental Health
Expression of ras (p21) protein in plasma from exposed workers and from patients with lung disease Diana Anderson Department of Biomedical Sciences, University of Bradford, Richmond Road, Bradford, West Yorkshire BD7 1DP
Abstract Oncogenes are involved with the regulation of cellular proliferation and thus could be important in the development of many cancers. Cells transformed in culture by ras genes can be activated either by the introduction of specific point mutations or by overexpression of the normal proto-oncogene. The ras genes encode a protein of 189 amino acids (molecular mass 21 kDa) designated as p21. ras p21 proteins are contained in all eukaryotic cells on the inner surface of the plasma membrane. We measured ras p21 proteins in lung cancer patients, patients with chronic obstructive pulmonary disease (COPD) and workers exposed to emissions from petrochemical plants and 1,3-butadiene and 1,3-butadiene/styrene. Proteins were separated by gel electrophoresis, transferred to a nitrocellulose membrane by Western blotting and detected by chemiluminescence. A monoclonal pan-ras antibody was used as the primary antibody. Optical densities of the peak area of the protein bands were calculated and values which were two standard deviations above negative control means were considered positive. Many of the cancer patients and some of the COPD patients gave positive responses, whilst exposed worker groups did not show statistically significant increases by comparison with the controls. Thus, an increase in ras oncoproteins could be a biomarker for cancer or the disease state in general, but it cannot be ruled out that it is a biomarker for exposure since many of the individuals examined were smokers exposed to cigarette smoke. Key words: ras (p21) proteins ± petroleum emissions ± 1,3-butadiene ± workers ± patients
Introduction Populations cannot be considered genetically homogeneous groups. However, susceptibility and realisation of disease outcome after exposure can be modified if repair is well-regulated and the steady state and balance of the individual occurs. When self-regulation fails, immunological responses can be connected to stress, depression and cancer. Oncogenes are involved with the regulation of cellular proliferation and thus could be important in the development of many cancers (Bishop, 1987; Kellen, 1995). Cells transformed in culture by ras genes can be activated either by the introduction of specific point mutations (e.g. at codons 12, 13, 61,
63 or 119) or by overexpression of the normal protooncogene (Barbacid, 1987; Perimental, 1989). The ras genes exert their effect in cells through the expression of their protein products, designated as p21. ras p21 proteins are contained in all eukaryotic cells on the inner surface of the plasma membrane (Barbacid, 1987; Perimental, 1989). Ras is frequently found to be activated in many human cancers (Bishop, 1991). Mutations in codons 12, 13 or 61 of the 3 ras genes, H-ras, K-ras and N-ras convert these genes into active oncogenes. Ras oncogenes can be found in a variety of tumour types, although the incidence varies greatly. The highest incidences are found in adenocarcinoma of the pancreas (90%), the colon (50%), the lung (30%), in thyroid tumours (50%) and in myeloid
Corresponding author: Diana Anderson, Department of Biomedical Sciences, University of Bradford, Richmond Road, Bradford, West Yorkshire BD7 1DP, Phone: 44 127 423 3569, Fax: 44 127 430 9742, E-mail: [email protected] 1438-4639/01/204-55 $ 15.00/0
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leukaemia (30%) (Bos, 1989). There is some correlation between tumour type and ras gene mutation. In adenocarcinoma of the lung, pancreas and colon, the K-ras gene is the predominantly expressed one. In order to further explore the value of the ras oncoprotein in plasma as a biomarker for the carcinogenic process, we have examined samples from untreated Polish human lung cancer patients and compared them with healthy donors. We have also compared Polish patients with COPD to the lung cancer patients to determine if ras oncoprotein increases can be a biomarker for the disease state in general. In addition, we have examined workers exposed to 1,3-butadiene and 1,3-butadiene/styrene from the Czech Republic and workers exposed to petroleum emissions from Poland and Estonia to help clarify whether increased ras oncoproteins are a biomarker of effect (the disease state) or exposure.
Materials and methods The plasma samples have been screened for the presence of ras p21 proteins using direct analysis of peripheral blood plasma based on the methods described earlier (Anderson et al., 1996a, b, c). Briefly, standard amounts of protein quantitated using the Lowry method (Lowry et al., 1951) were separated by gel electrophoresis, transferred to a nitrocellulose membrane by Western blotting and detected by chemiluminescence. A monoclonal ras antibody was used as the primary antibody and horse-radish peroxidase-conjugated sheep anti-mouse immunoglobulin as the secondary antibody. Optical densities (OD) of the peak of the protein bands were calculated and values of two standard deviations above negative control means were considered positive (Perera et al., 1992). There was also found to be a linear relationship between ODs and concentrations of ras (Anderson et al., 1996b). No attempt was made in the studies described to discriminate between the mutant proteins involved, merely to identify increased levels; therefore a pan-ras antibody was used which was able to detect proteins produced by all three members (Harvey, Kirsten and N-ras) of the ras gene family. Individuals examined Ten millilitre of heparinised peripheral blood samples were obtained from the different individuals and plasma was removed by centrifugation. Protease inhibitors and sodium dodecyl sulphate (SDS) were added and the mixture dispensed into aliquots and stored at ÿ 70 8C until required. Samples of 40 Polish lung cancer and 20 COPD patients and appropriate controls (35 and 20, respectively) were obtained by Drs E. Nizankowska and B. Graca (Collegium Medicum, Cracow, Poland). Samples of 25 Polish workers exposed to petroleum emissions and 35 controls were provided by Dr Antonina Cebulska-Wasilewska (Institute of Nuclear Physics, Cracow, Poland). Drs
T. Veidebaum (Estonian Institute of Experimental and Chemical Medicine, Tallina, Estonia), K. Peltonen and M. Sorsa (Finnish Institute of Occupational Health, Helsinki, Finland) provided samples from workers from a Czech plant near Prague exposed to 1,3-butadiene (10 individuals), 1,3-butadiene plus styrene (13 individuals) and 20 unexposed controls, and from workers from Estonia exposed at a petrochemical plant in the shale-oil area (97 workers: 50 exposed to benzene, 47 to polyaromatic hydrocarbons and benzene in a cokery) and 40 unexposed controls. All results were statistically analysed using a pooled two-sample t-test, untransformed and non-parametric Mann-Whitney test. Non-parametric statistics were used because there were a number of zero values in the control and exposed groups so violating a normal distribution. Lung cancer patients and controls The 40 untreated patients had an average age of 60.1 years and various occupations (actor, bricklayer, butcher, carpenter, chemist, clerk, driver, farmer, glider, hairdresser, librarian, locksmith, plumber, postman, shop assistant, teacher, turner etc.) (Anderson et al., 1996c). The 35 controls had a similar range of occupations and an average age of 40.2 years and were chosen randomly from Southern Poland (10 individuals), people working in offices in a petroleum factory (7 individuals) and from a region of Southern Poland with the lowest ratio of deaths from cancer to the overall death rate (18 individuals). COPD patients and their controls COPD is defined as a disease characterised by the presence of airflow obstruction due to bronchitis or emphysema and the diagnosis of COPD was established according to the criteria proposed by the American Thoracic Society (1995). The pulmonary function tests of the 20 COPD patients were: FVC (forced vital capacity) 1.46 l, i.e. 55% of predicted volume; FEV1 (forced expiratory volume on ls) 0.71 l, i.e. 35.4% of predicted volume; FEV1 % FVC 49.55%. The pulmonary function tests of their controls were FVC 3.92 l, i.e. 95.7% of predicted volume; FEV1 2.96 l, i.e. 89.1% of predicted volume; FEV1 % FVC 75.3%. The COPD patients had a mean age of 65.9 years and their negative controls of 62.4 years and were recruited from a similar area (Anderson et al., 1998).
Results Comparisons of lung cancer and COPD patients and controls by age Eighteen out of 40 (45%) lung cancer patients (mean age 60.1 years) had increased ras oncoprotein levels when compared to 35 concurrent controls (40.2 years) with a mean 2 SD of 2.53 (Table 1). Similarly, nine out of 20 (45%) COPD patients (65.9 years) had increased ras oncoprotein levels
Comparative ras (p21) protein studies in workers and patients
57
Table 1. Ras levels in plasma from cancer patients measured as the peak area ( O.D.) of bands at 21 kDa (150 mg protein, 2 min film exposure).
Workers exposed to 1,3-butadiene and 1,3-butadiene/styrene and controls in the Czech Republic
Sample number
Peak area
Sample number
Peak area
34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53
0 0 2.36 3.97a 0 1.87 4.06a 2.19 1.53 2.02 3.54a 1.75 2.06 1.84 3.76a 4.43a 2.69a 1.13 4.20a 2.01
54 55R 56R 57R 58R 59R 60R 61R 62R 63R 64R 67 68 69 70 71 72 73 74 75
2.94a 5.02a/5.3a M 5.16a 1.56/1.69 M 1.625 5.07a/5.41a M 5.24a 2.35/2.43 M 2.39 2.26/2.29 M 2.275 5.58a/5.79a M 5.685a 4.36a/4.78a M 4.57a 2.36/2.59a M 2.475 1.62/1.98 M 1.80 0/0 5.35a 3.47a 2.28 3.76a 3.69a 2.16 0.59 7.33a 4.66a
Similarly, there was one elevated level in each group by comparison with the mean 2 SD of the negative control group. There were no statistically significant differences between groups (Anderson et al., 1996b).
M mean of two results; R repeat exposure of same blot. a Peak area ( O.D.) higher than mean 2 S.D. of control group (> 2.53).
when compared to 20 age-matched controls (62.4 years) with a mean 2 SD of 0.70 (Table 2). However, 35 out of 40 (87.5%) lung cancer patients (60.1 years) had increased ras oncoprotein levels when compared to the 20 age-matched controls with a mean 2 SD of 0.70 (Anderson et al., 1996c, 1998). Comparisons of lung cancer and COPD patients with historical controls The lung cancer patients and COPD patients were compared with historical control values from plasma samples from all healthy control individuals from various countries (Table 3). 30 out of 40 (75%) lung cancer patients had increased ras oncoprotein levels and 4 out of 20 (20%) COPD patients had increased levels (Anderson et al., 1998). Workers exposed to emissions from petroleum plants and controls in Estonia The mean 2 SD of the control group gave a value of 1.50. This gave three elevated values in the exposed group compared with two in the control. There were no statistically significant differences between the groups (Anderson et al., 1987).
Workers exposed to emissions from petroleum plants in Poland There was a significant difference by comparison with the negative controls but this was due to the effects of smoking. In all of the above studies, samples in some instances were run on two separate gels, and results were reproducible. On other occasions samples were run at widely separated time intervals and results were also reproducible (Anderson et al., 1996a).
Discussion For lung cancer patients, whether using concurrent controls, age-matched controls or historical controls, an increase in ras p21 protein levels of lung cancer patients could be a possible prognostic biomarker for lung cancer (Anderson et al., 1998). Similarly for COPD patients, whether using agematched or historical controls, they had lower ras p21 protein values than cancer patients (Anderson et al., 1987). The ras p21 protein values of COPD patients might also be a biomarker for cancer. It is possible that some of these patients were in the process of developing cancer or perhaps would have died from COPD before the cancer was apparent. It cannot be ruled out that the increase in ras p21 values could be a biomarker of exposure since many of the lung cancer patients and most of the COPD patients were smokers (Anderson et al., 1987). As for the workers exposed to 1,3-butadiene, 1,3butadiene/styrene in the Czech Republic (Anderson et al., 1996b) and emissions from petroleum plants in Estonia (Anderson et al., 1987), there were increases in ras p21 values above control values but they were not statistically significant. This is in keeping with the negative findings for cytogenetics in these studies and low exposures in these plants. The significant increases in the ras p21 oncoproteins in the workers exposed in the Polish plant were found to be due to smoking (Anderson et al., 1996a). All these studies were carried out over a similar time frame, so the experimental system used is capable of detecting positive responses as witnessed
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D. Anderson
Table 2. Number coding of plasma samples, background information, clinical characteristics and ras p21 protein peak area values of patients with COPD and healthy volunteers from Poland. Number
Sex
Job
Age (y)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
M F F M F M M M M M M M M M M F M M M M M F M F M M M F F F M M M M M M M M M M
Construction engineer 58 Diet designer 60 Nurse 53 Economist 61 Diet designer 60 Worker 75 Librarian 76 Construction work 67 Mechanic engineer 65 Guardian 65 Mechanic engineer 55 Worker 76 Driver 59 Electrician 72 Nurse 51 Worker 55 Worker 69 Turner 60 Worker 76 Farmer 64 Worker 63 Dress maker 63 Worker 52 Housewife 63 Baker 78 Driver 73 Worker 70 Clerk 65 Chemical magazine worker 65 Housekeeper 74 Miner 66 Gas fitter 78 Blacksmith 81 Farmer 84 Farmer 73 Farmer 74 Farmer 70 Farmer 60 Farmer 69 Farmer 70 Mean 2 standard deviations
Diagnosis
Smoker
Cancer in family
Healthy 0 Y Healthy 0 Y Healthy 0 Y Healthy 1 N Healthy 0 Y Healthy 1/0 N Healthy 1 N Healthy 1/0 Healthy 0 Y Healthy 0 N Healthy 0 N Healthy 0 N Healthy 0 Y Healthy 0 Y Healthy 0 N Healthy 1 N Healthy 1 N Healthy 1 Y Healthy 1/0 N Healthy 1 N COPD 1/0 N COPD 1/0 N COPD 1/0 N COPD 1/0 N COPD 1/0 N COPD 1/0 Y COPD 1 N COPD 0 N COPD 0 N COPD 0 Y COPD 1/0 COPD 1/0 Y COPD 1/0 Y COPD 1/0 N COPD 1/0 N COPD 1 N COPD 1/0 N COPD 1 N COPD 1 N COPD 1/0 Y of POL 8 (samples 1 ± 20) 0.70
Peak areaa 0.34 0.34 0 0 0 0 0.75b 0.13 0.13 0.15 0.77/0.80 (0.79b ) 0.51/0.51 (0.51) 0/0 (0) 0/0 (0) 0.23/0.14 (0.19) 0.41/0 (0.21) 0.30/0.20 (0.25) 0.50/0.32 (0.41) 0.10/0.15 (0.13) 0/0 (0) 0 1.49b 0.43 4.34b 1.05b 0.38 2.82b 0.36 2.47b 0 0.15 0.50 0.59 1.98b 1.15b 0 0 0 1.56b 0.79b
COPD Chronic obstructive pulmonary disease according to American Thoracic Society criteria (1995); Y yes; N no; y years; Smoker status: 0 non-smoker; 1/0 former smoker; 1 current smoker; Underlined sample numbers the same blot was exposed twice for 2 min and the mean of the peak areas used. a Peak area of band at 21 kDa using samples containing 150 mg protein and 2 min film exposure. b Elevated levels of ras p21 protein by comparison with mean 2 SD of control mean of healthy volunteers ( POL 8).
by results from COPD and cancer patients. Other workers (Brandt-Rauf, 1991; Brandt-Rauf et al., 2000; Li et al., 1998; Luo et al., 1998) have found increased levels of ras oncoproteins in exposed workers. Since it has been reported that K-ras mutations are a relatively late event in the pathogenesis of any disease (Sugio et al., 1994) it might not be until the frank disease state is manifest, that increases can be detected. Also ras production may be primarily at the tumour site and restricted in the general circula-
tion. It has been suggested (Scambia et al., 1994) that it is not the amount of ras which is important in human pathogenesis, but whether or not a mutation has occurred (Slebos et al., 1990; Levine et al., 1994) and/or whether other mutations occur in oncogenes. The most common genetic alterations in human cancers involve the inhibitory function of the p53 tumour suppresser gene product (Levine et al., 1991). The p53 tumour suppresser gene can mutate to prevent tumour suppression or apoptosis (Malkin, 1994). In fact p53 protein expression has also
Comparative ras (p21) protein studies in workers and patients Table 3. Mean values of peak areas of ras p21 protein and plasma samples from all healthy control individuals obtained from various countries used to constitute historical mean value. Sample group
Number in group
Mean
SD
Mean 2 SD
POL 3 POL 5 POL 7 POL 8 EST 3 EST 6 SPAIN 2 Historical control mean
9 7 18 20 26 14 7 101
0.46 0.97 1.15 0.22 0.63 0.38 0.26 0.59
0.56 0.58 0.89 0.24 0.49 0.44 0.20 0.62
1.58 2.13 2.93 0.70 1.61 1.26 0.66 1.83
POL Polish samples; EST Estonian samples; SPAIN Spanish samples. POL 3 Unexposed people ± winter sampling from Southern Poland ( Anderson et al., 1996c); POL 5 Unexposed workers ± winter sampling from petrochemical plant in Southern Poland (Anderson et al., 1996c); POL 7 Unexposed people from a `clean' area of Southern Poland, i.e. a region of Poland showing the lowest ratio of deaths from cancer: deaths from other causes (Anderson et al., 1996c); POL 8 Healthy volunteer controls in this present study; EST 3 Unexposed people living in a rural area of Estonia ( Anderson et al., 1987); EST 6 Unexposed people also living in a rural area of Estonia ( Anderson et al., 1987); SPAIN 2 Unexposed people from Spain (unpublished).
been reported in vinyl chloride-exposed workers in Taiwan (Luo et al., 1999) in addition to Ki-ras oncoprotein expression (Luo et al., 1998). Perera (1997) discusses the role of environment and cancer and gives some insight into such interactions. Not all individuals get cancer. However in those that do, in addition to insult from carcinogens, the body's own defense mechanisms might break down. Mental depression, for example, can cause the release of adrenal corticoids causing immune suppression and decreasing lymphocyte proliferation. Such changes might ultimately trigger the cancer state particularly if repair mechanisms fail (FrentzelBeyme, 2000). Indirect effects such as a lack of well being might therefore be of critical importance in the development of cancer. Acknowledgements. The author would like to thank all the co-authors of the papers cited jointly in her papers in this communication for their contributions to the scientific work.
References American Thoracic Society. Definitions, epidemiology, pathophysiology, diagnosis and staging, American Journal of Respiratory Critical Care and Medicine 1995; 152: S78 ± S83. Anderson, D et al. Biological monitoring of workers exposed to emissions from petroleum plants, Environmental Health Perspectives 1996a; 104: 609 ± 613.
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Anderson, D et al. Examination of ras (p21) proteins in plasma from workers exposed to benzene emissions from petrochemical plants and healthy controls, Mutation Research 1987; 381: 149 ± 155. Anderson, D et al. Levels of ras oncoproteins in human plasma from 1,3-butadiene-exposed workers and controls, Mutation Research 1996b; 349: 115 ± 120. Anderson, D et al. Ras oncoproteins in human plasma from lung cancer patients and healthy controls, Mutation Research 1996c; 349: 121 ± 126. Anderson, D et al. Ras p21 protein levels in human plasma from patients with chronic obstructive pulmonary disease (COPD) compared with lung cancer patients and healthy controls, Mutation Research 1998; 403: 229 ± 235. Barbacid M. Ras genes, Annual Review of Biochemistry 1987; 56: 779 ± 827. Bishop JM. Molecular theories in oncogenesis, Cell 1991; 64: 235 ± 248. Bishop JM. The molecular genetics of cancer, Science 1987; 235: 305 ± 311. Bos JH. Ras oncogenes in human cancer: a review, Cancer Research 1989; 49: 4682 ± 4698. Brandt-Rauf PW. Advances in cancer biomarkers as applied to chemical exposures: the ras oncogene and p21 protein and pulmonary carcinogenesis, Journal of Occupational Medicine 1991; 33: 951 ± 955. Brandt-Rauf PW et al. Mutant oncoprotein biomarkers of vinyl chloride exposure: Applications to risk assessment. In: Andrson, D, Karakaya, AE, Sram, R, eds. Human monitoring after environmental and occupational exposure to chemical and physical agents. IOS Press The Netherlands 2000, pp 243 ± 248. Frentzel-Beyme, R. From host factors to biomarkers of susceptibility ± increased risk for cancer in persons with depressed mood. In: Anderson, D, Karakaya, AE, Sram, R, eds. Human monitoring after environmental and occupational exposure to chemical and physical agents. IOS Press The Netherlands 2000, pp. 213 ± 223. Kellen JA. Molecular interrelationships in multidrug resistance, Anticancer Research 1995; 14: 433 ± 435. Levine, AJ et al. The 1993 Walter Hubert lecture: the role of p53 tumour-suppressor gene in tumourigenesis, British Journal of Cancer 1994; 69: 409 ± 416. Levine, AJ et al. The p53 tumour suppressor gene, Nature 1991; 351: 453 ± 456. Li, Y et al. Mutant p21 ras in vinyl chloride-exposed workers, Biomarkers 1998; 3: 433 ± 439. Lowry, OH et al. Protein measurement with Folin phenol reagent, Journal of Biological Chemistry 1951; 193: 265 ± 275. Luo, JC et al. Plasma Asp13 Ki-ras oncoprotein expression in vinyl chloride monomer workers in Taiwan. Journal of Occupational and Environmental Medicine 1998; 40: 1053 ± 1058. Luo, JC et al. Plasma p53 protein and anti-p53 antibody expression in vinyl chloride monomer workers in Taiwan, Journal of Occupational and Environmental Medicine 1999; 41: 521 ± 526.
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Malkin, D. Germline p53 gene mutations and cancer ± Pandora's box or open sesame? Journal of the National Cancer Institute 1994; 86: 326 ± 328. Perera, FP et al. Molecular and genetic damage in humans from environmental pollution in Poland, Nature 1992; 360: 256 ± 258. Perera, FP. Environment and Cancer, who are susceptible? Science 1997; 278: 1068 ± 1073. Pimental E. Oncogenes, CRC Press, Boca Raton, FL 1989. Scambia, G et al. Expression of ras oncogene p21 protein in normal and neoplastic laryngeal tissues: correlation
with histopathological features and epidermal growth factor receptors, British Journal of Cancer 1994; 69: 995 ± 999. Slebos, RJC et al. K-ras oncogene activation as a prognostic marker in adenocarcinoma of the lung, New England Journal of Medicine 1990; 323: 561 ± 565. Sugio, K et al. K-ras mutations are a relatively late event in the pathogenesis of lung carcinomas, Cancer Research 1994; 54: 5811 ± 5815.
International Journal of Hygiene and Environmental Health
Expression of ras (p21) protein in plasma from exposed workers and from patients with lung disease Diana Anderson Department of Biomedical Sciences, University of Bradford, Richmond Road, Bradford, West Yorkshire BD7 1DP
Abstract Oncogenes are involved with the regulation of cellular proliferation and thus could be important in the development of many cancers. Cells transformed in culture by ras genes can be activated either by the introduction of specific point mutations or by overexpression of the normal proto-oncogene. The ras genes encode a protein of 189 amino acids (molecular mass 21 kDa) designated as p21. ras p21 proteins are contained in all eukaryotic cells on the inner surface of the plasma membrane. We measured ras p21 proteins in lung cancer patients, patients with chronic obstructive pulmonary disease (COPD) and workers exposed to emissions from petrochemical plants and 1,3-butadiene and 1,3-butadiene/styrene. Proteins were separated by gel electrophoresis, transferred to a nitrocellulose membrane by Western blotting and detected by chemiluminescence. A monoclonal pan-ras antibody was used as the primary antibody. Optical densities of the peak area of the protein bands were calculated and values which were two standard deviations above negative control means were considered positive. Many of the cancer patients and some of the COPD patients gave positive responses, whilst exposed worker groups did not show statistically significant increases by comparison with the controls. Thus, an increase in ras oncoproteins could be a biomarker for cancer or the disease state in general, but it cannot be ruled out that it is a biomarker for exposure since many of the individuals examined were smokers exposed to cigarette smoke. Key words: ras (p21) proteins ± petroleum emissions ± 1,3-butadiene ± workers ± patients
Introduction Populations cannot be considered genetically homogeneous groups. However, susceptibility and realisation of disease outcome after exposure can be modified if repair is well-regulated and the steady state and balance of the individual occurs. When self-regulation fails, immunological responses can be connected to stress, depression and cancer. Oncogenes are involved with the regulation of cellular proliferation and thus could be important in the development of many cancers (Bishop, 1987; Kellen, 1995). Cells transformed in culture by ras genes can be activated either by the introduction of specific point mutations (e.g. at codons 12, 13, 61,
63 or 119) or by overexpression of the normal protooncogene (Barbacid, 1987; Perimental, 1989). The ras genes exert their effect in cells through the expression of their protein products, designated as p21. ras p21 proteins are contained in all eukaryotic cells on the inner surface of the plasma membrane (Barbacid, 1987; Perimental, 1989). Ras is frequently found to be activated in many human cancers (Bishop, 1991). Mutations in codons 12, 13 or 61 of the 3 ras genes, H-ras, K-ras and N-ras convert these genes into active oncogenes. Ras oncogenes can be found in a variety of tumour types, although the incidence varies greatly. The highest incidences are found in adenocarcinoma of the pancreas (90%), the colon (50%), the lung (30%), in thyroid tumours (50%) and in myeloid
Corresponding author: Diana Anderson, Department of Biomedical Sciences, University of Bradford, Richmond Road, Bradford, West Yorkshire BD7 1DP, Phone: 44 127 423 3569, Fax: 44 127 430 9742, E-mail: [email protected] 1438-4639/01/204-55 $ 15.00/0
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D. Anderson
leukaemia (30%) (Bos, 1989). There is some correlation between tumour type and ras gene mutation. In adenocarcinoma of the lung, pancreas and colon, the K-ras gene is the predominantly expressed one. In order to further explore the value of the ras oncoprotein in plasma as a biomarker for the carcinogenic process, we have examined samples from untreated Polish human lung cancer patients and compared them with healthy donors. We have also compared Polish patients with COPD to the lung cancer patients to determine if ras oncoprotein increases can be a biomarker for the disease state in general. In addition, we have examined workers exposed to 1,3-butadiene and 1,3-butadiene/styrene from the Czech Republic and workers exposed to petroleum emissions from Poland and Estonia to help clarify whether increased ras oncoproteins are a biomarker of effect (the disease state) or exposure.
Materials and methods The plasma samples have been screened for the presence of ras p21 proteins using direct analysis of peripheral blood plasma based on the methods described earlier (Anderson et al., 1996a, b, c). Briefly, standard amounts of protein quantitated using the Lowry method (Lowry et al., 1951) were separated by gel electrophoresis, transferred to a nitrocellulose membrane by Western blotting and detected by chemiluminescence. A monoclonal ras antibody was used as the primary antibody and horse-radish peroxidase-conjugated sheep anti-mouse immunoglobulin as the secondary antibody. Optical densities (OD) of the peak of the protein bands were calculated and values of two standard deviations above negative control means were considered positive (Perera et al., 1992). There was also found to be a linear relationship between ODs and concentrations of ras (Anderson et al., 1996b). No attempt was made in the studies described to discriminate between the mutant proteins involved, merely to identify increased levels; therefore a pan-ras antibody was used which was able to detect proteins produced by all three members (Harvey, Kirsten and N-ras) of the ras gene family. Individuals examined Ten millilitre of heparinised peripheral blood samples were obtained from the different individuals and plasma was removed by centrifugation. Protease inhibitors and sodium dodecyl sulphate (SDS) were added and the mixture dispensed into aliquots and stored at ÿ 70 8C until required. Samples of 40 Polish lung cancer and 20 COPD patients and appropriate controls (35 and 20, respectively) were obtained by Drs E. Nizankowska and B. Graca (Collegium Medicum, Cracow, Poland). Samples of 25 Polish workers exposed to petroleum emissions and 35 controls were provided by Dr Antonina Cebulska-Wasilewska (Institute of Nuclear Physics, Cracow, Poland). Drs
T. Veidebaum (Estonian Institute of Experimental and Chemical Medicine, Tallina, Estonia), K. Peltonen and M. Sorsa (Finnish Institute of Occupational Health, Helsinki, Finland) provided samples from workers from a Czech plant near Prague exposed to 1,3-butadiene (10 individuals), 1,3-butadiene plus styrene (13 individuals) and 20 unexposed controls, and from workers from Estonia exposed at a petrochemical plant in the shale-oil area (97 workers: 50 exposed to benzene, 47 to polyaromatic hydrocarbons and benzene in a cokery) and 40 unexposed controls. All results were statistically analysed using a pooled two-sample t-test, untransformed and non-parametric Mann-Whitney test. Non-parametric statistics were used because there were a number of zero values in the control and exposed groups so violating a normal distribution. Lung cancer patients and controls The 40 untreated patients had an average age of 60.1 years and various occupations (actor, bricklayer, butcher, carpenter, chemist, clerk, driver, farmer, glider, hairdresser, librarian, locksmith, plumber, postman, shop assistant, teacher, turner etc.) (Anderson et al., 1996c). The 35 controls had a similar range of occupations and an average age of 40.2 years and were chosen randomly from Southern Poland (10 individuals), people working in offices in a petroleum factory (7 individuals) and from a region of Southern Poland with the lowest ratio of deaths from cancer to the overall death rate (18 individuals). COPD patients and their controls COPD is defined as a disease characterised by the presence of airflow obstruction due to bronchitis or emphysema and the diagnosis of COPD was established according to the criteria proposed by the American Thoracic Society (1995). The pulmonary function tests of the 20 COPD patients were: FVC (forced vital capacity) 1.46 l, i.e. 55% of predicted volume; FEV1 (forced expiratory volume on ls) 0.71 l, i.e. 35.4% of predicted volume; FEV1 % FVC 49.55%. The pulmonary function tests of their controls were FVC 3.92 l, i.e. 95.7% of predicted volume; FEV1 2.96 l, i.e. 89.1% of predicted volume; FEV1 % FVC 75.3%. The COPD patients had a mean age of 65.9 years and their negative controls of 62.4 years and were recruited from a similar area (Anderson et al., 1998).
Results Comparisons of lung cancer and COPD patients and controls by age Eighteen out of 40 (45%) lung cancer patients (mean age 60.1 years) had increased ras oncoprotein levels when compared to 35 concurrent controls (40.2 years) with a mean 2 SD of 2.53 (Table 1). Similarly, nine out of 20 (45%) COPD patients (65.9 years) had increased ras oncoprotein levels
Comparative ras (p21) protein studies in workers and patients
57
Table 1. Ras levels in plasma from cancer patients measured as the peak area ( O.D.) of bands at 21 kDa (150 mg protein, 2 min film exposure).
Workers exposed to 1,3-butadiene and 1,3-butadiene/styrene and controls in the Czech Republic
Sample number
Peak area
Sample number
Peak area
34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53
0 0 2.36 3.97a 0 1.87 4.06a 2.19 1.53 2.02 3.54a 1.75 2.06 1.84 3.76a 4.43a 2.69a 1.13 4.20a 2.01
54 55R 56R 57R 58R 59R 60R 61R 62R 63R 64R 67 68 69 70 71 72 73 74 75
2.94a 5.02a/5.3a M 5.16a 1.56/1.69 M 1.625 5.07a/5.41a M 5.24a 2.35/2.43 M 2.39 2.26/2.29 M 2.275 5.58a/5.79a M 5.685a 4.36a/4.78a M 4.57a 2.36/2.59a M 2.475 1.62/1.98 M 1.80 0/0 5.35a 3.47a 2.28 3.76a 3.69a 2.16 0.59 7.33a 4.66a
Similarly, there was one elevated level in each group by comparison with the mean 2 SD of the negative control group. There were no statistically significant differences between groups (Anderson et al., 1996b).
M mean of two results; R repeat exposure of same blot. a Peak area ( O.D.) higher than mean 2 S.D. of control group (> 2.53).
when compared to 20 age-matched controls (62.4 years) with a mean 2 SD of 0.70 (Table 2). However, 35 out of 40 (87.5%) lung cancer patients (60.1 years) had increased ras oncoprotein levels when compared to the 20 age-matched controls with a mean 2 SD of 0.70 (Anderson et al., 1996c, 1998). Comparisons of lung cancer and COPD patients with historical controls The lung cancer patients and COPD patients were compared with historical control values from plasma samples from all healthy control individuals from various countries (Table 3). 30 out of 40 (75%) lung cancer patients had increased ras oncoprotein levels and 4 out of 20 (20%) COPD patients had increased levels (Anderson et al., 1998). Workers exposed to emissions from petroleum plants and controls in Estonia The mean 2 SD of the control group gave a value of 1.50. This gave three elevated values in the exposed group compared with two in the control. There were no statistically significant differences between the groups (Anderson et al., 1987).
Workers exposed to emissions from petroleum plants in Poland There was a significant difference by comparison with the negative controls but this was due to the effects of smoking. In all of the above studies, samples in some instances were run on two separate gels, and results were reproducible. On other occasions samples were run at widely separated time intervals and results were also reproducible (Anderson et al., 1996a).
Discussion For lung cancer patients, whether using concurrent controls, age-matched controls or historical controls, an increase in ras p21 protein levels of lung cancer patients could be a possible prognostic biomarker for lung cancer (Anderson et al., 1998). Similarly for COPD patients, whether using agematched or historical controls, they had lower ras p21 protein values than cancer patients (Anderson et al., 1987). The ras p21 protein values of COPD patients might also be a biomarker for cancer. It is possible that some of these patients were in the process of developing cancer or perhaps would have died from COPD before the cancer was apparent. It cannot be ruled out that the increase in ras p21 values could be a biomarker of exposure since many of the lung cancer patients and most of the COPD patients were smokers (Anderson et al., 1987). As for the workers exposed to 1,3-butadiene, 1,3butadiene/styrene in the Czech Republic (Anderson et al., 1996b) and emissions from petroleum plants in Estonia (Anderson et al., 1987), there were increases in ras p21 values above control values but they were not statistically significant. This is in keeping with the negative findings for cytogenetics in these studies and low exposures in these plants. The significant increases in the ras p21 oncoproteins in the workers exposed in the Polish plant were found to be due to smoking (Anderson et al., 1996a). All these studies were carried out over a similar time frame, so the experimental system used is capable of detecting positive responses as witnessed
58
D. Anderson
Table 2. Number coding of plasma samples, background information, clinical characteristics and ras p21 protein peak area values of patients with COPD and healthy volunteers from Poland. Number
Sex
Job
Age (y)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
M F F M F M M M M M M M M M M F M M M M M F M F M M M F F F M M M M M M M M M M
Construction engineer 58 Diet designer 60 Nurse 53 Economist 61 Diet designer 60 Worker 75 Librarian 76 Construction work 67 Mechanic engineer 65 Guardian 65 Mechanic engineer 55 Worker 76 Driver 59 Electrician 72 Nurse 51 Worker 55 Worker 69 Turner 60 Worker 76 Farmer 64 Worker 63 Dress maker 63 Worker 52 Housewife 63 Baker 78 Driver 73 Worker 70 Clerk 65 Chemical magazine worker 65 Housekeeper 74 Miner 66 Gas fitter 78 Blacksmith 81 Farmer 84 Farmer 73 Farmer 74 Farmer 70 Farmer 60 Farmer 69 Farmer 70 Mean 2 standard deviations
Diagnosis
Smoker
Cancer in family
Healthy 0 Y Healthy 0 Y Healthy 0 Y Healthy 1 N Healthy 0 Y Healthy 1/0 N Healthy 1 N Healthy 1/0 Healthy 0 Y Healthy 0 N Healthy 0 N Healthy 0 N Healthy 0 Y Healthy 0 Y Healthy 0 N Healthy 1 N Healthy 1 N Healthy 1 Y Healthy 1/0 N Healthy 1 N COPD 1/0 N COPD 1/0 N COPD 1/0 N COPD 1/0 N COPD 1/0 N COPD 1/0 Y COPD 1 N COPD 0 N COPD 0 N COPD 0 Y COPD 1/0 COPD 1/0 Y COPD 1/0 Y COPD 1/0 N COPD 1/0 N COPD 1 N COPD 1/0 N COPD 1 N COPD 1 N COPD 1/0 Y of POL 8 (samples 1 ± 20) 0.70
Peak areaa 0.34 0.34 0 0 0 0 0.75b 0.13 0.13 0.15 0.77/0.80 (0.79b ) 0.51/0.51 (0.51) 0/0 (0) 0/0 (0) 0.23/0.14 (0.19) 0.41/0 (0.21) 0.30/0.20 (0.25) 0.50/0.32 (0.41) 0.10/0.15 (0.13) 0/0 (0) 0 1.49b 0.43 4.34b 1.05b 0.38 2.82b 0.36 2.47b 0 0.15 0.50 0.59 1.98b 1.15b 0 0 0 1.56b 0.79b
COPD Chronic obstructive pulmonary disease according to American Thoracic Society criteria (1995); Y yes; N no; y years; Smoker status: 0 non-smoker; 1/0 former smoker; 1 current smoker; Underlined sample numbers the same blot was exposed twice for 2 min and the mean of the peak areas used. a Peak area of band at 21 kDa using samples containing 150 mg protein and 2 min film exposure. b Elevated levels of ras p21 protein by comparison with mean 2 SD of control mean of healthy volunteers ( POL 8).
by results from COPD and cancer patients. Other workers (Brandt-Rauf, 1991; Brandt-Rauf et al., 2000; Li et al., 1998; Luo et al., 1998) have found increased levels of ras oncoproteins in exposed workers. Since it has been reported that K-ras mutations are a relatively late event in the pathogenesis of any disease (Sugio et al., 1994) it might not be until the frank disease state is manifest, that increases can be detected. Also ras production may be primarily at the tumour site and restricted in the general circula-
tion. It has been suggested (Scambia et al., 1994) that it is not the amount of ras which is important in human pathogenesis, but whether or not a mutation has occurred (Slebos et al., 1990; Levine et al., 1994) and/or whether other mutations occur in oncogenes. The most common genetic alterations in human cancers involve the inhibitory function of the p53 tumour suppresser gene product (Levine et al., 1991). The p53 tumour suppresser gene can mutate to prevent tumour suppression or apoptosis (Malkin, 1994). In fact p53 protein expression has also
Comparative ras (p21) protein studies in workers and patients Table 3. Mean values of peak areas of ras p21 protein and plasma samples from all healthy control individuals obtained from various countries used to constitute historical mean value. Sample group
Number in group
Mean
SD
Mean 2 SD
POL 3 POL 5 POL 7 POL 8 EST 3 EST 6 SPAIN 2 Historical control mean
9 7 18 20 26 14 7 101
0.46 0.97 1.15 0.22 0.63 0.38 0.26 0.59
0.56 0.58 0.89 0.24 0.49 0.44 0.20 0.62
1.58 2.13 2.93 0.70 1.61 1.26 0.66 1.83
POL Polish samples; EST Estonian samples; SPAIN Spanish samples. POL 3 Unexposed people ± winter sampling from Southern Poland ( Anderson et al., 1996c); POL 5 Unexposed workers ± winter sampling from petrochemical plant in Southern Poland (Anderson et al., 1996c); POL 7 Unexposed people from a `clean' area of Southern Poland, i.e. a region of Poland showing the lowest ratio of deaths from cancer: deaths from other causes (Anderson et al., 1996c); POL 8 Healthy volunteer controls in this present study; EST 3 Unexposed people living in a rural area of Estonia ( Anderson et al., 1987); EST 6 Unexposed people also living in a rural area of Estonia ( Anderson et al., 1987); SPAIN 2 Unexposed people from Spain (unpublished).
been reported in vinyl chloride-exposed workers in Taiwan (Luo et al., 1999) in addition to Ki-ras oncoprotein expression (Luo et al., 1998). Perera (1997) discusses the role of environment and cancer and gives some insight into such interactions. Not all individuals get cancer. However in those that do, in addition to insult from carcinogens, the body's own defense mechanisms might break down. Mental depression, for example, can cause the release of adrenal corticoids causing immune suppression and decreasing lymphocyte proliferation. Such changes might ultimately trigger the cancer state particularly if repair mechanisms fail (FrentzelBeyme, 2000). Indirect effects such as a lack of well being might therefore be of critical importance in the development of cancer. Acknowledgements. The author would like to thank all the co-authors of the papers cited jointly in her papers in this communication for their contributions to the scientific work.
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Anderson, D et al. Examination of ras (p21) proteins in plasma from workers exposed to benzene emissions from petrochemical plants and healthy controls, Mutation Research 1987; 381: 149 ± 155. Anderson, D et al. Levels of ras oncoproteins in human plasma from 1,3-butadiene-exposed workers and controls, Mutation Research 1996b; 349: 115 ± 120. Anderson, D et al. Ras oncoproteins in human plasma from lung cancer patients and healthy controls, Mutation Research 1996c; 349: 121 ± 126. Anderson, D et al. Ras p21 protein levels in human plasma from patients with chronic obstructive pulmonary disease (COPD) compared with lung cancer patients and healthy controls, Mutation Research 1998; 403: 229 ± 235. Barbacid M. Ras genes, Annual Review of Biochemistry 1987; 56: 779 ± 827. Bishop JM. Molecular theories in oncogenesis, Cell 1991; 64: 235 ± 248. Bishop JM. The molecular genetics of cancer, Science 1987; 235: 305 ± 311. Bos JH. Ras oncogenes in human cancer: a review, Cancer Research 1989; 49: 4682 ± 4698. Brandt-Rauf PW. Advances in cancer biomarkers as applied to chemical exposures: the ras oncogene and p21 protein and pulmonary carcinogenesis, Journal of Occupational Medicine 1991; 33: 951 ± 955. Brandt-Rauf PW et al. Mutant oncoprotein biomarkers of vinyl chloride exposure: Applications to risk assessment. In: Andrson, D, Karakaya, AE, Sram, R, eds. Human monitoring after environmental and occupational exposure to chemical and physical agents. IOS Press The Netherlands 2000, pp 243 ± 248. Frentzel-Beyme, R. From host factors to biomarkers of susceptibility ± increased risk for cancer in persons with depressed mood. In: Anderson, D, Karakaya, AE, Sram, R, eds. Human monitoring after environmental and occupational exposure to chemical and physical agents. IOS Press The Netherlands 2000, pp. 213 ± 223. Kellen JA. Molecular interrelationships in multidrug resistance, Anticancer Research 1995; 14: 433 ± 435. Levine, AJ et al. The 1993 Walter Hubert lecture: the role of p53 tumour-suppressor gene in tumourigenesis, British Journal of Cancer 1994; 69: 409 ± 416. Levine, AJ et al. The p53 tumour suppressor gene, Nature 1991; 351: 453 ± 456. Li, Y et al. Mutant p21 ras in vinyl chloride-exposed workers, Biomarkers 1998; 3: 433 ± 439. Lowry, OH et al. Protein measurement with Folin phenol reagent, Journal of Biological Chemistry 1951; 193: 265 ± 275. Luo, JC et al. Plasma Asp13 Ki-ras oncoprotein expression in vinyl chloride monomer workers in Taiwan. Journal of Occupational and Environmental Medicine 1998; 40: 1053 ± 1058. Luo, JC et al. Plasma p53 protein and anti-p53 antibody expression in vinyl chloride monomer workers in Taiwan, Journal of Occupational and Environmental Medicine 1999; 41: 521 ± 526.
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