Putrescine, DNA, RNA and protein contents in human uterine, breast and rectal cancer.M Bandopadhyay, AK Ganguly
Department of Medicine, University College of Medicine, Dr. B. C. Roy Postgraduate Institute of Basic Medical Sciences, Calcutta-700 020, India., India
Correspondence Address: Source of Support: None, Conflict of Interest: None PMID: 11298463
Source of Support: None, Conflict of Interest: None
AIMS: To find out the status of DNA, RNA and protein in human uterine, ovarian, breast and rectal carcinoma. MATERIAL AND METHODS: In this prospective study, patients of age group between late thirties and late fifties suffering from uterine, ovarian, breast and rectal cancer were taken as subjects of the present study. The total number of cases studied for each cases was ten. Pieces of human carcinomatous tissues of above mentioned cases were taken along with surrounding normal tissues. From the tissue samples, putrescine is separated by the method of Herbst et al, DNA analysed by Diphenylamine method, RNA by Orcinol method and protein by Biuret method. RESULTS: Tissue content of putrescine rises simultaneously with that of DNA, RNA and protein in carcinomatous growths as above in comparison to their respective adjacent normal tissue, the differences being statistically highly significant. CONCLUSIONS: Increase in DNA, RNA and protein concentration may be a pre-requisite for increased synthesis of putrescine in carcinomatous tissue and thereby the concentration of other di- and poly-amines.
Keywords: Adult, Biopsy, Needle, Breast Neoplasms, chemistry,Comparative Study, DNA, Neoplasm, analysis,Female, Human, Male, Middle Age, Oncogene Proteins, analysis,Ovarian Neoplasms, chemistry,Probability, Prospective Studies, Putrescine, analysis,RNA, Neoplasm, analysis,Rectal Neoplasms, chemistry,Sensitivity and Specificity, Spectrophotometry, Support, Non-U.S. Gov′t, Tissue Culture, Tumor Markers, Biological, analysis,Uterine Neoplasms, chemistry,
It is well known that polyamines and cellular growths are involved in growth processes.,,, From the results of an elegant series of experiments by various investigators, it has been revealed that increased intracellular polyamine concentration has an intimate relation with increased rate of cell proliferation and actively dividing mammalian cells contain higher levels of polyamines than quiscent cells.,,,
General stimulation of protein synthesis is required for the cellular growth, and neoplastic growth is always associated with increased DNA synthesis., Inhibitor of ornithine decarboxylase (ODC; 188.8.131.52) is known to be a rate-limiting enzyme in the polyamine biosynthetic pathway,, such as alpha-difluoromethylorn-ithine (DFMO) influences the cytotoxicity of antitumour agents by interacting with DNA has been demonstrated. Exogenous spermidine could restore both RNA levels and spermidine/spermine N1-acetyltransferase (N1SSAT) activity in polyamine depleted cells.
In view of the controversies and the various forms of observations, it was thought that it will be important to elucidate whether protein, DNA and RNA contents change along with polyamine (putrescine) concentration in different carcinomatous growths.
This prospective study was carried out from October 1998 to February 2000. Pieces of human uterine, ovarian, breast and rectal carcinomatous tissues were collected, from Gyneocology and Surgery Department of PG Hospital, Calcutta, along with surrounding normal tissues (which were confirmed on histology to be normal), removed during surgery. These were taken in separate vials (Borosil), labelled and preserved in a deep fridge at –260C.
Putrescine was separated by high voltage electrophoresis in a Durram type electrophoresis cabinet with a regulated power supply by the method of Herbst et al. The tissue samples were weighed dry, chilled on ice, cut into fine pieces, homogenised in 0.1 N HCl, extracted in butanol and then separated by high voltage electrophoresis. The samples were applied 10 cm from the end on the anode side of the paper strip by streaking with a 25 ?l micropipette. The buffer system used for this particular experiment was 0.03 M citric acid at pH 6.5. After the run at a potential of 300V the strips were removed and excess buffer on the ends of the strips blotted to prevent any distortion of pattern. The paper strips were dried in an oven at 1000C, stained by 0.25% ninhydrin in acetone solution and returned to the oven for developing colour.
The colour developed was read at 570 m? in a spectrophotometer and the readings obtained were plotted on the standard curves of putrescine to get the corresponding quantitative value, expressed in ?mol/g of tissue. Putrescine was obtained from the Sigma Chemical Co, St. Louis, USA.
Protein, DNA and RNA concentrations were determined by spectrophotometric methods. Tissues were homogenised with cold 0.25 M sucrose. DNA analysis was done by Diphenylamine method, RNA by Orcinol method, and protein by Biuret method. All the chemicals used in this study were of analytical grade. Results were expressed as means ? SEM. A paired t-test was used to determine the significance of the data obtained. p values less than 0.05 were considered as significant.
A total of ten cases in each of the four carcinomatous groups, viz. uterine, ovarian, breast and rectal carcinoma, were inclued in the study. The values of putrescine, DNA and RNA in these groups in the carcinomatous tissue and the adjacent normal tissue has been presented in 4 tables. The first one [Table - 1] shows the putresine concentrations in tissues of uterine, ovarian, breast and rectal cancer patients as compared to that of adjacent normal tissues, while the second table [Table - 2] presents DNA concentrations in tissues of the same group of patients as compared to the same in adjacent normal tissues. In [Table - 3], RNA concentrations have been shown in tissues of the same carcinomatous growths in comparison to their surrounding normal tissues. The protein concentrations in carcinomatous growths under study have also been presented in [Table - 4] along with the concentrations of the same in adjacent normal tissues.
ODC has been shown to be involved only in differentiation of cells and not in proliferation. Reports exist to show metastasis of cancer is inhibited by injection of putrescine., HeLa cell proliferation was found to be increased with addition of diamine oxidase (DAO; EC 184.108.40.206) in tissue culture experiments, although DAO degrades biogenic amines released from cells; whereas HeLa cell proliferation was decreased by iproniazid which maintains high polyamine levels. Polyamine biosynthesis inhibitor can selectively suppress putrescine without affecting spermidine and spermine of experimental breast cancer in rats. Further, polyamine deprivation prevents the development of tumour-induced immune-suppression. We have demonstrated, however, an increase in polyamine concentrations in carcinomatous tissues as well as in serum and urine in a number of carcinomatous growths investigated so far.,,,
It has been revealed from various studies that polyamines are short chain aliphatic amines required for normal cellular growth that are ubiquitously found in all living tissues. In our previous experiments with different carcinomatous tissues we demonstrated significant elevation of diamines and polyamines and also in DAO activity confirming their positive correlation with neoplastic growths.,,,
It has been also been shown that in the absence of defined regulators (growth factors or hormones), certain amino acids may contribute to the epigenetic control of human tumour cell invasion and their metastasis. Changes in cellular polyamine levels may affect the degree of DNA methylation. It has been reported that a putrescine level with a cut off point of 1.5 nmol/mg protein is the most accurate single discriminator of risk status in familial adenomatous polyposis.
It is clear from this experiment that not only putrescine concentration increases in uterine, ovarian, breast and rectal carcinoma tissues in comparison to their respective control values, but also the concentrations of DNA, RNA and protein increase unequivocally in the above carcinomatous growths. It might be speculated that due to the general stimulation of protein synthesis there is increased synthesis of ODC which is involved in the synthesis of polyamines. It appears therefore, that increase in DNA, RNA and protein concentration is a pre-requisite for increased synthesis of putrescine in carcinomatous tissues and thereby the concentrations of other di and polyamines, in view of putrescine being the mother substance of them.
The authors gratefully acknowledge the financial assistance given by the University Grants Commission, Government of India, for carrying out this investigation.
[Table - 1], [Table - 2], [Table - 3], [Table - 4]