[Prognostic value of erythrocyte polyamines levels in renal cell carcinoma. Prospective study in 418 cases]. / Valeur pronostique des polyamines érythrocytaires dans le cancer du rein. Etude chez 418 patients.

OBJECTIVES: Polyamines: Spermine (Spm) and Spermidine (Spmd), are essential for cell proliferation and differentiation. A measurement of erythocytes polyamines (EPA) was developed in our institution. Our objective was to evaluate this marker as a new prognostic factor in renal cell carcinoma. PATIENTS AND METHODS: A blood sample was prospectively taken before surgery, among 418 patients who had an enlarged nephrectomy (n=318) or a partial nephrectomy (n=100) to quantify EPA rates by using the HPLC technique. The qualitative and quantitative variables have been compared using chi(2) and Student statistical analyses. The survivals have been normalized by the Kaplan Meier and Cox methods. RESULTS: The average age of our population was 64 years (21-88). The average decline was 41 months (1-214). The median size of tumors was 6.5cm (1-24). The median rate of Spm and Spmd were respectively 4.7 (1-83) and 9 (2-86)nmol/8.10(9) erythrocytes. Spm and Spmd were linked to the T stage (p=0.001), and the ECOG (p=0.001 and 0,008). Spm was not linked at N and M stages but at the Fuhrman grade (p=0.001). Spmd was linked to the N, M stages (p=0.04). With univariate analysis, the tumor diameter, the TNM stage, the Fuhrman grade as well as Spm and Spmd (p<0.0001) were predictors of specific survival. With multivariate analysis, some prognostic factors remained independent: the TNM stage, the ECOG and Spmd, a continuous variable (p=0.0001), pushing the rank of Fuhrman out of the model. When Spm and Spmd were dichotomized in quantitative variables, they were both independent factors. CONCLUSION: The EPA is a new prognostic tool, before surgery, which will be tested for its integration into prognostic normograms.

Polyamine contents in current foods: a basis for polyamine reduced diet and a study of its long term observance and tolerance in prostate carcinoma patients.

Polyamine contents were assessed by mass spectrometry in 233 current foods and beverages. In order to reduce gut polyamine uptake, a polyamine reduced diet (PRD) and partial intermittent intestinal tract decontamination (PIITD) with neomycin or nifuroxazide was proposed as nutritional therapy to 33 prostate carcinoma patients, 30 of whom with hormone refractory prostate cancer (HRPC). Mean PRD observance was 22 +/- 19 (median: 16; range: 3-72) months. 10, 8 and 3 patients were respectively on PRD for more than 30, 36 and 64 months. No diet toxicity was observed. 8 patients had moderate intestinal intolerance due to PIITD which was interrupted. No significant differences in body weight, blood counts or serum protein levels were observed during the follow-up of patients under PRD. Performance status and pain scores were relatively stable during the trial with improved pain scores at 6 months. A PRD associated with intermittent PIITD is a safe and well observed nutritional regimen and long term observance is possible.

Contralateral effect of amitriptyline and bupivacaine for sciatic nerve block in an animal model of inflammation.

BACKGROUND: Using a carrageenan inflammation rat model, we evaluated two experimental approaches to prolong sciatic nerve block on contralateral hyperalgesia. Method. We performed ipsilateral sciatic nerve block on the inflamed hind paw with bupivacaine-loaded microspheres suspended in dexamethasone (bupivacaine 12.5 mg) and with amitriptyline (6.25 and 12.5 mg) as ultralong-acting local anaesthetics. Bupivacaine (1.25 mg) was used as long-acting local anaesthetic and saline was used as a control. The sixth group received amitriptyline 6.25 mg intraperitoneally (n=10 for each group). RESULTS: The duration of ipsilateral nerve block was 2 h for bupivacaine, 7 h for amitriptyline 6.25 mg, 11 h for amitriptyline 12.5 mg and 21 h for bupivacaine-loaded microspheres in suspension with dexamethasone. Whereas contralateral hyperalgesia was not observed during block produced by bupivacaine-loaded microspheres, contralateral hyperalgesia was observed with sciatic nerve block using amitriptyline. CONCLUSIONS: Because of the differential effect observed on the contralateral side, the mechanism underlying the prolongation of ipsilateral block with amitriptyline may not result only from a prolonged Na(+) channel blockade but might be explained by a local toxic effect or lack of systemic actions.

Polyamine-reduced diet in metastatic hormone-refractory prostate cancer (HRPC) patients.

Polyamine (PA) deprivation is effective in prostate carcinoma models. We have assessed the observance by patients, tolerance and side effects of a PA-reduced diet (PRD) and intestinal decontamination (ID), in order to reduce PA dietary and intestinal bacterial pools, in metastatic, hormone-refractory prostate cancer (HRPC) patients. A total of 13 volunteers (mean age, 67+/-10 years) with metastatic HRPC were proposed for PRD and ID (0.75 g/day of oral neomycin every other week). The mean time from HRPC diagnosis to the start of the diet was 12+/-8 months. Of the total 13, seven patients had received prior chemotherapy or Estramustine phosphate. PRD was obtained after HPLC assessment of PA contents in current foods and given 5 days a week. Toxicity, performance and pain status were assessed according to the World Health Organisation and EORTC scales. Prostatic specific antigen (PSA), blood counts, ionograms, transaminases and erythrocyte PA spermidine (Spd) and spermine (Spm; assessed by HPLC) were evaluated regularly. Mean observance was 8+/-7 months (range, 2-26 months). One case of grade II toxicity to neomycin was observed. Cancer-specific survival (after the diet) was 14+/-7 months, and two patients are still alive. All the other patients have died of their cancer at 12+/-6 months (range, 4-20 months). Cancer-specific survival after hormonal escape was 27+/-11 months (range, 9-45 months). Performance status was improved during the regimen and deteriorated 3 months after stopping. Pain score was improved (1.3 versus 0.6; P =0.04) during the diet and increased (2.1 versus 0.3) 3 months after stopping. Erythrocyte Spd (11.6+/-7 versus 7.7+/-2 nmol/8 x 10(9) erythrocytes; P =0.036) and Spm (7+/-6 versus 3.9+/-1.6 nmol/8 x 10(9) erythrocytes; P =0.036) levels were significantly reduced at 3 months. One patient had a >50% reduction in PSA, three patients had PSA stabilization for 6 months. PSA progression was observed in all other patients. No significant modification of other studied biological parameters was noted. Reducing PA dietary intake and ID is a well-observed and tolerated regimen and seems to be beneficial for patient quality of life and pain control. Patients with low initial PSA can experience durable stabilization. These encouraging results in such an aggressive disease certainly warrant further investigation.

Dimethylsilane polyamines: cytostatic compounds with potentials as anticancer drugs. II. Uptake and potential cytotoxic mechanisms.

Dimethylsilane tetramines are structural analogues of spermine with a (CH3)2 Si-group incorporated into the central carbon chain. They have potential as anticancer drugs. Their cytotoxic effect was considered to rely mainly on their polyamine antagonist property. In order to obtain new ideas about cellular mechanisms, which are potential targets of the dimethylsilane polyamines, the effects of these compounds on some basic cell functions, such as protein and DNA synthesis, and calmodulin antagonism were studied. In addition, their mode of accumulation in cells was investigated. It became evident that the intracellular accumulation of dimethylsilane polyamines is almost exclusively achieved via the polyamine transport system. However, the exchange of a part of the intracellular natural polyamines against dimethylsilane polyamines has only a small effect on polyamine uptake. Binding to the endoplasmic reticulum and inhibition of protein synthesis are presumably important for the cytotoxic action of bis(11-amino-4,8-diazaundecyl)dimethylsilane, a hexamine, but seem of no importance for the tetramines. Calmodulin antagonism, however, is likely to contribute to their cytotoxic effect.

Clinical significance of cell proliferation, microvessel density, and CD44 adhesion molecule expression in renal cell carcinoma.

Renal cell carcinoma (RCC) is known to have a wide variation in clinical outcome despite the use of conventional prognostic factors, such as staging or grading. A better knowledge of the biologic aggressiveness of RCC could facilitate the selection of patients at high risk of tumor progression. The aim of this study was to determine if use of measurements of vascular density, cell proliferation, and cell adhesion could better predict the biologic behavior of RCC. We immunohistochemically analyzed CD34, Ki-67, and CD44H expression on formalin-fixed, paraffin-embedded tissues from 73 RCCs for quantifying microvessel density (MVD), Ki-67 labeling index (LI), and CD44H LI, respectively. Univariate cancer-specific survival analysis showed that tumor stage (P < .01), tumor size (P < .001), nuclear grade (P < .01), metastasis (P < .001), MVD (P < .03), Ki-67 LI (P < .001), and CD44H LI (P < .0001) were predictors of tumor-related death. There was a statistical correlation between CD44H LI and both Ki-67 LI (r' = .3) and MVD (r' = -44). Ki-67 LI (P < .04) and CD44H LI (P < .02), as well as metastasis (P < .008), emerged as independent predictors of cancer-specific survival in multivariate analysis in patients with metastases (P < .04 and P < .02, respectively) and in patients without metastases (P < .006 and P < .00001, respectively). Our study suggests that vascular density, cell proliferation, and cell adhesion represent a complex tumor-host interaction that may favor progression of RCC. Cell proliferation and CD44H expression appear to be powerful markers to identify patients with an adverse prognosis.

N-Benzylpolyamines as vectors of boron and fluorine for cancer therapy and imaging: synthesis and biological evaluation.

Cancer cells have high-affinity polyamine uptake systems with a low stringency for structural features. Putrescine, spermidine, and spermine have, therefore, been considered as potential vectors for the selective accumulation in tumors of therapeutically or diagnostically useful structures and elements. We envisaged N-benzyl derivatives of the polyamines as vectors of (10)B and (18)F for boron neutron capture therapy (BNCT) and tumor imaging by positron emission tomography (PET), respectively. In the present work, the synthesis, transport characteristics, DNA-binding properties, and cytotoxicity of several N-benzyl derivatives of putrescine and spermidine are described. The fluorinated spermidine derivative N-(3-[(4-aminobutyl)amino]propyl)[(4-fluorophenyl)methyl]amine (N(1)-4-Fbz-spd) may be useful for PET because of its high accumulation in cancer cells via the polyamine transport system. Among the boron-containing benzyl polyamines, N-(4-aminobutyl)([4-(dihydroxyboryl)phenyl]methyl)amine (4-Bbz-put) and N-(3-[(4-aminobutyl)amino]propyl)([4-(dihydroxyboryl)phenyl]methyl)amine (N(1)-4-Bbz-spd) should be suitable for BNCT, because their accumulation in B16 melanoma cells was more efficient than that of borocaptate and borophenylalanine, two reference compounds used in BNCT.

Polyamine profiles in tumor, normal tissue of the homologous breast, blood, and urine of breast cancer sufferers.

Polyamines are involved in the development of breast cancer. We assayed polyamines in erythrocytes, urines, and breast tissues (tumor tissue and histologically normal breast tissue close to the tumor) of patients with invasive breast cancer (n = 174) and benign breast disease (n = 71, used as controls). Polyamine levels in red blood cells and urine were similar to the polyamine concentrations found in healthy subjects, and thus cannot be used as diagnostic markers of breast cancer. In cancer tissue, polyamines were significantly increased in comparison with the polyamine concentrations in controls, and were correlated to the tumor aggressiveness as evaluated by histological grade and Ki-67 proliferative index. On the other hand, correlation was found between polyamine levels in the tumor and the status of the hormone receptors. In the mammary tissue close to the cancer, polyamines dramatically decreased in comparison with the polyamine levels of tissue samples removed around the histologically proven benign tumors. The changes of the polyamine concentrations in the histologically normal breast tissue in the vicinity of the cancer could play a role in the cancer development and need further studies, especially if polyamines are considered as a potential therapeutic target in breast cancer.

Polyamines are unevenly distributed within the rat and rabbit kidney.

Aliphatic polyamines have generally been measured on the whole kidney. Since the kidney is composed of a variety of cells, whole organ data are of limited value for the interpretation of the functions of the polyamines. The aim of this study was to establish the distribution pattern of putrescine, spermidine and spermine within the kidneys of male and female rats and rabbits. It is shown that the polyamines are unevenly distributed along the cortico-papillary axis. Each amine exhibited its own distinct distribution pattern. The polyamines are predominantly located in the cortex. Putrescine levels increased gradually from the cortex to the papillary tip in rabbits, whereas, in rats, fluctuations in putrescine level were marked. In the six zones of the rabbit kidney studied, spermidine and spermine concentrations were markedly higher in females than in males. This difference was less marked in rats.

Benefits of complete polyamine deprivation in hormone responsive and hormone resistant MCF-7 human breast adenocarcinoma in vivo.

Polyamines are ubiquitous cellular compounds which are required for estradiol induced proliferation in breast cancer. Complete polyamine deprivation, using 2 alpha-difluoromethyl-ornithine (DFMO, Eflornithine), a specific inactivator of ornithine decarboxylase (key-enzyme of the polyamine biosynthesis) combined with inhibition of the bacterial production of gastrointestinal polyamine and a polyamine free regimen, was demonstrated to exhibit a cytostatic effect and a decrease of the three tumoral polyamine concentrations in a MCF-7 tumor model. In this experiment, complete polyamine deprivation has been tested on a standard MCF-7 tumor and on a variant MCF-7 tumor (i.e. with a tamoxifen acquired resistance). Polyamine deprivation was effective on the tumor growth, both on standard and variant sub-types. The polyamine contents of two types of tumor were similar, and identically, polyamine deprivation has caused a decrease of putrescine, spermidine and also spermine tumoral concentrations measured by the HPLC method in standard and variant MCF-7 tumors. Acquired tamoxifen resistance is common in patients undergoing hormonal therapy for advanced breast cancer. It has been hypothesized that the direct stimulation of polyamine pathway without estradiol involvement could be one of the mechanisms responsible for the tamoxifen resistance. The ability of polyamine deprivation to inhibit the growth of tumors becoming tamoxifen resistant could offer a therapeutic advantage in case of tumor with acquired tamoxifen resistance and could be tested to prevent or delay the hormonal responsiveness to breast cancer.

Value of immunohistochemical Ki-67 and p53 determinations as predictive factors of outcome in renal cell carcinoma.

OBJECTIVES: Nuclear grade and tumor stage are important prognostic factors in renal cell carcinoma, but tumors of similar stage and grade can exhibit a wide variation in biologic behavior and clinical outcome. In this retrospective study, we evaluated the immunologic markers, Ki-67 (MIB1) and p53, in 73 cases of conventional (clear cell) renal cell carcinoma and compared these markers with the accepted prognostic features of grade, stage, and tumor size in predicting outcome. METHODS: Specimens of 73 renal cell carcinomas of different nuclear grade (20 Furhman I/II, 32 Fuhrman III, and 21 Fuhrman IV) and different stage (10 pT1, 23 pT2, 36 pT3, and 4 pT4) were immunostained with monoclonal antibodies against Ki-67 and p53. RESULTS: Univariate statistical analysis showed that tumor size (P <0. 001), nuclear grade (P <0.01), tumor stage (P <0.01), Ki-67 index (P <0.001), and p53 immunostaining (P <0.03) correlated significantly with a poor prognosis. A Ki-67 index of 20% was a powerful predictor of survival in all patients (P <0.00001), with strong predictive values. On multivariate analysis, the Ki-67 index and metastases were significant independent prognostic factors (P <0.02 and <0.01, respectively). CONCLUSIONS: Ki-67 immunostaining appeared to be an additional prognostic indicator of biologic aggressiveness in renal cell carcinoma. Immunohistochemical assessment of tumor antigens could be used to identify patients at high risk of tumor progression in addition to conventional prognostic factors.

Polyamine deprivation alters formalin-induced hyperalgesia and decreases morphine efficacy.

Although the exact functions of polyamines in the nervous system remain still unclear, they are thought to have a physiological role in intracellular signal processing and neurotransmission. Polyamine deprivation which consists in the reduction of both the endogenous and exogenous sources of polyamines is a promising treatment for cancer. In a previous study we have shown that this treatment provokes an analgesic effect in rats submitted to brief phasic nociceptive tests. The present study examined the effect of polyamine deprivation on pain-related behaviors and spinal c-fos expression evoked in the formalin test presumed to better reflect clinical pain, using morphine as analgesia control. Polyamine deprivation per se altered the characteristic pain-related behaviors, reducing the interphase depression of pain, without inducing changes in the spinal Fos staining. In addition this treatment prevented the antinociceptive effect of morphine both on behavioral responses and on spinal c-fos expression. In polyamine-deprived rats, despite morphine injection, nociceptive scores remained dramatically high during the intermediate and the late phases of the response and the number of Fos immunoreactive neurons remained largely higher in deeper layers than in morphine control rats. Altogether these data support a modulatory role of polyamines both on the neuronal circuitry mediating sensory information, and on mechanisms underlying morphine analgesia.

Polyamines in human breast cancer and its relations to classical prognostic features: clinical implications.

Experimental evidence suggest an important role of polyamines in breast cancer development. Polyamines have been determined in tissue and erythrocyte samples from 100 patients with primary invasive breast cancer and 30 patients with fibroadenomas. Statistical analysis was performed in order to determine the prognostic value of the polyamine patterns of tumor tissues and erythrocytes in comparison with clinical and histological prognostic factors. In malignant tissues, polyamine levels were significantly higher than in benign tissues. They correlated with markers of tumor aggressivity (axillary node involvement and especially with markers of high mitotic rate as Ki-67 staining, histological grade). No correlation was found between estrogen and progesterone status, tumor size and polyamine concentrations. Erythrocyte polyamines levels were identical between cancer patients and controls. The knowledge of the polyamine pattern in breast cancer could become useful in clinical practice particularly if polyamine metabolism is targeted as a therapeutic approach.

Induction of Fos protein expression in spinal cord neurons of tumour-bearing rats.

The absence of discernible abnormal symptoms such as pain, often leading to delayed diagnosis in cancer patients, may be indicative of a dysregulation in sensory transmission between the tumour and the central nervous system. We explored expression of Fos protein in spinal cord neurons in rats, during the development of the MAT-LyLu prostatic adenocarcinoma grafted on the hind limb. The tumour triggered the densest Fos labelling in the L3-L5 lumbar segments, ipsilateral to the grafted limb. The labelling, detected at day 5, increased until day 10 and dropped off thereafter. The ventral horn (except lamina IX) was the most densely labelled region. Histological examination of the grafted limbs demonstrated that no inflammatory reaction accompanied the tumour growth. Rats exhibited no behavioural alterations either spontaneous or induced by handling. These results demonstrate that signals are sent to the central nervous system by the peripheral tumour. Considering both the behavioural and histological observations, it is unlikely that spinal activity reflects a painful state. The nature of these signals, inefficient to trigger the appropriate reaction of the organism against the tumour, remain to be determined with regard to the pharmacologically active compounds synthesized and released by the tumour cells.

Molecular analysis of the combining site of a monoclonal antibody against spermine.

The structural basis of the binding of the polyamine spermine to the monoclonal antibody SPM8-2 was studied using computer modelling, ELISA methods and chemical modifications of the binding site residues. Paratope modelling showed that the antibody combining site forms a highly negatively charged cavity mainly shaped by aspartic acid and tyrosine residues which contact the tetra-positively charged spermine molecule by electrostatic interactions and hydrogen bondings. The importance of the electrostatic environment for spermine binding to SPM8-2 is emphasised by the strong dependency on pH and ionic strength. Specific chemical modifications of carboxylate groups and tyrosine residues of the antibody adsorbed to microtiter plates resulted in decreased binding of the N1-biotin-spermine conjugate used to monitor the activity of the antibody. These observations are consistent with a key role of aspartate and tyrosine residues in complex formation with spermine. These studies, important to our understanding of antibody-hapten specificity, may also shed light on important motifs responsible for protein-polyamine interactions.

The gastrointestinal polyamine source depletion enhances DFMO induced polyamine depletion in MCF-7 human breast cancer cells in vivo.

Due to the polyamine requirement for cell growth, blockade of polyamine biosynthesis is considered a potential anticancer target. The lack of efficacy of DFMO in vivo, has been attributed to other sources of polyamines, mainly from the gastrointestinal tract (alimentary and bacterial). An experiment was designed to test the role of intestinal polyamine deletion in addition to DFMO (a specific inhibitor of ODC) in established MCF-7 tumors in nude mice. Using DFMO and polyamine-free diet, the tumor putrescine concentrations were more profoundly decreased in comparison to DFMO alone and cellular spermine was also depleted, as has never been observed with DFMO alone. The blockade of the gastrointestinal sources of polyamines enhances the intracellular polyamine depletion induced by DFMO on MCF-7 tumor in nude mice.

Polyamine sulfonamides with NMDA antagonist properties are potent calmodulin antagonists and cytotoxic agents.

N1-Dansylspermine and related sulfonamides of the natural polyamines are very potent blockers of NMDA-type glutamate receptors. They exhibit pharmacological properties which were not predicted from the constituents of the conjugates. Cytotoxicity and calmodulin antagonism of N1-dansylspermine were especially impressive. Calmodulin antagonism implies that N1-dansylspermine prevents induction of ornithine decarboxylase and inhibits its own active uptake via the polyamine transport system. Structure-activity considerations demonstrated that an aromatic character of the substituent is not required; amide bond formation with an aliphatic sulfonic acid is sufficient to transform spermine into a highly toxic calmodulin antagonist. Cytotoxicity and calmodulin antagonism are properties which are intrinsic to spermine, but they are observed only at very high concentrations. Amide bond formation at N1 with a lipophilic residue appears to 'amplify' these normally latent properties. The use of polyamine conjugates structurally related to the amides described in this work for targeting tumours may be marred by their calmodulin antagonism.

Tumour growth modifies intravascular polyamine transport by plasma lipoproteins in the mouse.

Polyamines are polycationic compounds which are implicated in cell division and tumor growth. We have evaluated the potential role of plasma lipoproteins in the transport of major polyamines, spermine, spermidine and putrescine, and the effect of tumor growth on such transport. Plasmas of healthy male BL6/DBA2 mice and of mice bearing Lewis lung carcinoma (3LL) were fractionated by isopycnic density gradient ultracentrifugation, and polyamine content determined in lipoprotein fractions. Spermidine was the most abundant polyamine in the lipoproteins of both control and tumor-bearing mice and was principally associated with HDL (d: 1.046-1.136 g/ml); approx. 40% of total plasma polyamines was lipoprotein-associated in control mice and 60% in cancerous mice. Only minor amounts were transported by LDL (< 10% of total lipoprotein-associated polyamines), while VLDL were devoid of these substances. Marked elevations of circulating levels of LDL were found in 3LL grafted mice: in these particles however, the contents of spermidine and spermine were significantly reduced. A preferential uptake of polyamines by red blood cells could in part explain this marked reduction of LDL polyamine content, but the consequence of this reduction on the net electrical charge and biochemical function of LDL remains unknown. Elevations of plasma LDL and HDL levels in 3LL-grafted mice underlie the finding that only minor modification was detected in the putrescine content of these particles. However, it is evident that elevated total amounts of putrescine were present in the plasma of such animals. Finally, the density profile of polyamines was modified in cancerous mice in which a shift to transport in lighter apo.AI-containing HDL particles was observed for spermidine; an even more marked shift was found for spermine. In conclusion, our data demonstrate that HDL particles constitute the major plasma vehicle for polyamine transport in both control and in tumor-bearing mice.

Flow cytometric analysis of in vivo polyamine deprivation in Lewis lung carcinoma (3LL) cells using the monoclonal antibody SPM8-2.

It has previously been shown that the monoclonal antibody SPM8-2 recognizes free spermine and spermidine as well as polyamines bound by an amide bond. In the present work it is demonstrated that this antibody also interacts with spermidine, spermine, and to a lesser extent N1- and N8-acetyl spermidine in an ELISA test where the polyamines are bound by reaction with formaldehyde. 3LL Lewis lung carcinoma cells from tumor-grafted mice were labeled with fluorescein-conjugated monoclonal antibody SPM8-2 and analyzed by flow cytometry. Both viable cells and formaldehyde-fixed and subsequently permeabilized cells showed fluorescent staining. However, most polyamines present in the cells are not directly available for antibody binding. Treatment of fixed cells with DNase or RNase greatly increased fluorescent staining, suggesting that some polyamines are co-localized with DNA and RNA. Antibody labeling of the cells was prevented by addition of free spermine. 3LL cells from tumors of mice treated by a polyamine depleting regimen had decreased intracellular spermidine levels and bound less antibody when compared to untreated controls. After digestion with RNase, the cells from treated mice bound considerably less fluorescent antibody than tumor cells from untreated mice, while their RNA content was similar. In contrast, fluorescent staining after DNase digestion was only slightly affected by the treatment with a polyamine depleting regimen. This suggests that the pools of polyamines which are co-localized with RNA are depleted more readily than those associated with DNA. Since only a small proportion of the intracellular polyamines is accessible to the bulky antibodies, treatment with hydrolytic enzymes (DNase, RNase) is necessary to reveal specific compartments of the polyamines and to demonstrate qualitative and semi-quantitative differences of their distribution within cells.