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 ::  Abstract
 ::  Material and method
 ::  Results
 ::  Discussion
 ::  Acknowledgment
 ::  References
 ::  Article Tables

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ORIGINAL ARTICLE
Year : 2000  |  Volume : 46  |  Issue : 3  |  Page : 172-5

Putrescine, DNA, RNA and protein contents in human uterine, breast and rectal cancer.


Department of Medicine, University College of Medicine, Dr. B. C. Roy Postgraduate Institute of Basic Medical Sciences, Calcutta-700 020, India., India

Correspondence Address:
M Bandopadhyay
Department of Medicine, University College of Medicine, Dr. B. C. Roy Postgraduate Institute of Basic Medical Sciences, Calcutta-700 020, India.
India
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Source of Support: None, Conflict of Interest: None


PMID: 11298463

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 :: Abstract 

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,


How to cite this article:
Bandopadhyay M, Ganguly A K. Putrescine, DNA, RNA and protein contents in human uterine, breast and rectal cancer. J Postgrad Med 2000;46:172

How to cite this URL:
Bandopadhyay M, Ganguly A K. Putrescine, DNA, RNA and protein contents in human uterine, breast and rectal cancer. J Postgrad Med [serial online] 2000 [cited 2019 Nov 18];46:172. Available from: http://www.jpgmonline.com/text.asp?2000/46/3/172/286


It is well known that polyamines and cellular growths are involved in growth processes.[1],[2],[3],[4] 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.[5],[6],[7],[8]

General stimulation of protein synthesis is required for the cellular growth[9],[10] and neoplastic growth is always associated with increased DNA synthesis.[11],[12] Inhibitor of ornithine decarboxylase (ODC; 4.1.1.17) is known to be a rate-limiting enzyme in the polyamine biosynthetic pathway,[13],[14] such as alpha-difluoromethylorn-ithine (DFMO) influences the cytotoxicity of antitumour agents by interacting with DNA has been demonstrated.[15] Exogenous spermidine could restore both RNA levels and spermidine/spermine N1-acetyltransferase (N1SSAT) activity in polyamine depleted cells.[16]

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.


  ::   Material and method Top


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.[17] 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,[18] RNA by Orcinol method,[19] and protein by Biuret method.[20] 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.




  ::   Results Top


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.




  ::   Discussion Top


ODC has been shown to be involved only in differentiation of cells and not in proliferation.[21] Reports exist to show metastasis of cancer is inhibited by injection of putrescine.[22],[23] HeLa cell proliferation was found to be increased with addition of diamine oxidase (DAO; EC 1.4.3.6) 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.[24] Polyamine biosynthesis inhibitor can selectively suppress putrescine without affecting spermidine and spermine of experimental breast cancer in rats.[25] Further, polyamine deprivation prevents the development of tumour-induced immune-suppression.[26] 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.[27],[28],[29],[30]

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.[31] 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.[27],[28],[29],[30]

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.[32] Changes in cellular polyamine levels may affect the degree of DNA methylation.[33] 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.[34]

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.[35] 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.




  ::   Acknowledgment Top


The authors gratefully acknowledge the financial assistance given by the University Grants Commission, Government of India, for carrying out this investigation.

 
 :: References Top

1. Cohen SS. Polyamines as a growth industry. Fed Proc 1982; 14: 3061-3064.  Back to cited text no. 1    
2.Oshawa N. Recent progress in polyamine research. Hum Cell 1990; 3:91-98.  Back to cited text no. 2    
3.Tabor CW, Tabor H. Polyamines. Annu Rev Biochem 1984; 53: 749-790.  Back to cited text no. 3    
4.Minuk GY, Bennaroch A, Ding LX. Polyamine transport system in isolated rat hepatocytes derived from resting and regenerating livers. Am J Physiol 1992; 263:G169-173.  Back to cited text no. 4    
5.Stjernborg L, Heby O, Mamont P, Persson L. Polyamine mediated regulation of S-adenosylmethionine expression in mammalian cells. Studies using 5’([(Z)-4-amino-2-butenyl] methylamino)-5’-deoxyadenosine, a suicide inhibitor of the enzyme. Eur J Biochem 1993; 214:671-676.  Back to cited text no. 5    
6.Cipolla B, Guille F, Moulinoux JP, Quemener V, Staerman F, Corbel L, et al. Polyamines and prostatic carcinoma: clinical and therapeutic implications. Eur Urol 1993; 24:124-131.  Back to cited text no. 6    
7.Heby O. Role of polyamines in the control of cell proliferation and differentiation. Differentiation 1981; 19:1-20.  Back to cited text no. 7    
8.Verma DS, Sunkara PS. An essential role for polyamine biosynthesis during granulopoetic differentiation. Cancer Res 1982; 42: 3046-3049.  Back to cited text no. 8    
10.Wang B, Soule HD, Miller FR. Transforming and oncogenic potential of activated c-Ha-ras in three immortalized human breast epithelial cell lines. Anticancer Res 1997; 17:4387-4394.  Back to cited text no. 10    
11.Nerlich AG, Wiest I, Wagner E, Sauer U, Schleicher ED. Gene expression and protein deposition of major basement membrane components and TGF-beta 1 in human breast cancer. Anticancer Res 1997; 7:4443-4449.  Back to cited text no. 11    
12.Basu HS, Pellarin M, Feuerstein BG, Shirahata A, Samejima K, Deen DF, et al. Introduction of a polyamine analogue, 1, 19-bis (ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4), with DNA and effect on growth, survival and polyamine levels in seven human brain tumour cell lines. Cancer Res 1993; 53:3948-3955.  Back to cited text no. 12    
13.Heby O. DNA methylation and polyamines in embryonic development and cancer. Int J Dev Biol 1995; 39:737-757.  Back to cited text no. 13    
14.Russell DH. Ornithine decarboxylase: a key regulatory enzyme in normal and neoplastic growths. Drug Metab Rev 1985; 16:1-88.  Back to cited text no. 14    
15.Manni A, Wechter R, Wei L, Heitjan D, Demers L. Phenotypic features of breast cancer cells overexpressing ornithine decarboxylase. J Cell Physiol 1995; 163:129-136.  Back to cited text no. 15    
16.Desiderio MA, Bergamaschi D, Mascellani E, De-Feudis P, Erba D’Incalci M. Treatment with inhibitors of polyamine biosynthesis which selectively lower intracellular spermine, does not affect the activity of alkylating agents but antagonizes the cytotoxicity of DNA-topois-omerase II inhibitors. Br J Cancer 1997; 75:1028-1034.  Back to cited text no. 16    
17.Ignatenko NA, Gerner EW. Growth arrest and polyamine-dependent expression of spermidine/spermine N1-acetyl-transferase in human tumour cells. Cell Growth Differ 1996; 7:481-486.  Back to cited text no. 17    
18.Herbst EJ, Keister DL, Weaver RH. Separation of Aliphatic Amines by paper chromatography or paper electrophoresis. Arch Biochem Biophys 1958; 75:178-185.  Back to cited text no. 18    
19.Dische Z, Schwarz K. In methods of Biochemical Analysis. 1954; Inter Science Publishers Inc., New York p.299.  Back to cited text no. 19    
20.Brown AM. In methods of Biochemical Analysis. 1954; Inter Science Publishers Inc: New York p.298.  Back to cited text no. 20    
21.Rabinovitz M, Olson ME, Greenberg DM. Methods of Biochemical Analysis. J Biol Chem 1954; 210:837  Back to cited text no. 21    
22.Nichols WK and Poser FH. Ornithine decarboxylase and differentiation of cells. Life Sci 1980; 27:1913-1920.  Back to cited text no. 22    
23.Boggust WA, O’Connell S, Carroll R, Wilson P. Inhibition of metastatic tumour developed in WHT/HE mice by putrescine, spermidine and spermine administration. I.R.C.S Med. Sci Compend 1980; 8:597-598.  Back to cited text no. 23    
24.Parchment RE. The implications of a unified theory of programmed cell death, polyamines, oxyradicals and histogenesis in the embryo. Int J Dev Biol 1993; 37:75-83.  Back to cited text no. 24    
25.Boggust WA. Changes in polyamine concentration in relation to the proliferation of HeLa cells. IRCS Libr Compend 1980; 8:600-601.  Back to cited text no. 25    
26.Manni A, Badger B, Glikman P, Bartholomew M, Santner S, Demers L. Individual and combined effects of alpha-difluoromethylornithine and ovariectomy on the growth and polyamine milieu of experimental breast cancer in rats. Cancer Res 1989; 49:3529-34.  Back to cited text no. 26    
27.Chamaillard L, Catros-Quemener V, Delcros JG, Bansard JY, Havouis R, Desury D, et al. Polyamine deprivation prevents the development of tumour-induced immunesuppression. Br J Cancer 1997; 76:365-70.  Back to cited text no. 27    
28.Chanda R, Ganguly AK. Diamine oxidase activity and tissue histamine content of human skin, breast and rectal carcinoma. Cancer Letters 1987; 34:207-212.  Back to cited text no. 28    
29.Chanda R, Ganguly AK. Polyamines in relation to human breast, rectal and squamous cell carcinoma. Cancer Letters 1988; 39:311-318.  Back to cited text no. 29    
30.Chanda R, Ganguly AK. Diamine oxidase activity and tissue di and polyamine contents of human ovarian, cervical and endometrial carcinoma. Cancer Letters 1995; 89:23-28.  Back to cited text no. 30    
31.Bandyopadhyay M, Chanda R, Ganguly AK. Putrescine, GABA profile in human breast, rectal, ovarian, cervical and endometrial carcinoma. Med Sci Res 1999; 27:49-51.  Back to cited text no. 31    
32.Wang W, Liu LQ, Higuchi CM. Mucosal polyamine measurements and colorectal cancer risk. J Cell Biochem 1996; 63:252-257.  Back to cited text no. 32    
33.Singh RK, Rinehart CA, Kim JP, Tolleson - Rinehart S, Lawing LF, Kaufman BG, et al. Tumour cell invasion of basement membrane in vitro is regulated by amino acids. Cancer Invest 1996; 14:6-18.  Back to cited text no. 33    
34.Hickok NJ, Uitto J. Regulation of ornithine decarboxylase gene expression polyamine levels and DNA synthetic rate by all-trans-retinoic acids in cultured human keratinocytes. J Invest Dermatol 1992; 98:327-32.  Back to cited text no. 34    
35.Giardiello FM, Hamilton SR, Hylind LM, Yang VW, Tamez P, Casero RA. Ornithine decarboxylase and polyamines in familial adenomatous polyposis. Cancer Res 1997; 57:199-201.  Back to cited text no. 35    
36.Linsalata M, Russo F, Cavallini A, Berloco P, Di-Leo A. Polyamines diamine oxidase and ornithine decarboxylase activity in colorectal cancer and in normal surrounding mucosa. Dis Colon Rectum 1993; 36:662-667.   Back to cited text no. 36    


    Tables

[Table - 1], [Table - 2], [Table - 3], [Table - 4]

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