Walker Carcinoma Research Articles

Stimulatory effects of amphetamine on the development of Walker-256 carcinoma lung metastases in rats

Experiments were performed in order to evaluate the effects of amphetamine (0.4 mg/kg/day) on the development of lung metastases in rats injected i.v. with 10(4) Walker 256 (W-256) carcinosarcoma cells, The number of metastatic nodules on the surface of the lungs, as well as the percentage-area of metastases in the frontal section through pulmonary hilus were increased in rats injected with amphetamine in comparison with those injected with placebo. Survival periods were also assessed and amphetamine was found to increase the lethality of rats.

1,25 Dihydroxyvitamin D and Dexamethasone DecreaseIn VivoWalker Carcinoma Growth, But Not Parathyroid Hormone Related Protein Secretion

Parathyroid hormone related protein (PTHrP) is produced by several breast cancers. 1,25 dihydroxyvitamin D (1,25[OH]2D) and Dexamethasone (DEX) have been shown to decrease PTHrP mRNA expression in several cell lines. We therefore tested the in vivo effect of both steroids on PTHrP secretion and tumor development of the Walker carcinoma (WC). WC cells were injected subcutaneously in Fisher rats which were simultaneously treated with either vehicle, or 1,25(OH)2D (0.5 micrograms/kg/d) or DEX (2 mg/kg/d). After 7 days, tumor weight was significantly decreased in the 2 treated-groups as compared to the control group. Vehicle treated-rats developed hypercalcemia, which was also observed in rats treated with 1,25(OH)2D; by contrast, the plasma calcium was significantly decreased in the DEX-treated group compared to vehicle-treated rats. In a dose-effect experiment, this dose of 1,25(OH)2D induced marked hypercalcemia in rats not implanted with WC, but was required to decrease the tumor weight in implanted rats. In both 1,25(OH)2D and DEX-treated groups, plasma PTHrP levels were significantly decreased, but there was a similar correlation between PTHrP plasma level and tumor weight in the three groups. Indeed, the cytosolic PTHrP content/mg tumor was identical in the 3 groups. By contrast, the PTHrP/Actin mRNA in the tumor was significantly decreased in the 1,25(OH)2D group, comparatively to the vehicle and DEX groups. Our results show that Dexamethasone and 1,25(OH)2D decrease WC tumor development in vivo, but do not change the PTHrP secretion by the remaining tumor although steady state PTHrP mRNA content level is decreased by 1,25(OH)2D.

Osteoporosis-like changes in Walker carcinoma 256-bearing rats, not accompanied with hypercalcemia or parathyroid hormone-related protein production.

Walker carcinoma 256 (W256) was reported to induce hypercalcemia dependent on bone metastasis and/or parathyroid hormone‐related protein (PTHrP) in the rat, providing a model of the humoral hypercalcemia of malignancy. In this study, after the subcutaneous inoculation of cells of the W256/S line, which is maintained in this laboratory, into young female Wistar Imamichi rats (6 weeks old), serum calcium and phosphorus levels changed only within the control range, whereas serum alkaline phosphatase activity and urinary calcium level significantly increased and urinary phosphorus decreased during the tumor growth, resulting in hypercalciuria and hypophosphaturia. W256/S did not express PTHrP‐mRNA, whereas LLC‐W256 cells did express it. Serum PTHrP level was not changed in W256/S‐bearing rats. Osteoporosis‐like changes, bone weight loss, low contents of bone calcium and phosphorus, and a decrease in the bone mineral density (BMD), were observed in the femur 14 days after the tumor inoculation. There was a pronounced decrease in the serum 17β‐estradiol level during the tumor growth. The reduction of BMD of femurs in W256/S‐bearing rats was significantly inhibited by treatment with salmon calcitonin or 17β‐estradiol. On the basis of these results, W256/S carcinoma‐bearing rats seem to be a useful model for osteoporosis of hypoovarianism.

Genotoxic effects of 3-amino-1,2,4-benzotriazine-1, 4-dioxide (SR 4233) and nitrogen mustard- N-oxide (nitromin) in Walker carcinoma cells under aerobic and hypoxic conditions

As judged by alkaline elution, exposure of Walker cells to either 3-amino-1,2,4-benzotriazine-1, 4-dioxide (SR 4233) or nitromin results in a dose-dependent increase in DNA damage due to single-strand breaks. With nitromin or SR 4233 there was little difference in the extent of DNA single-strand breaks between Walker cells incubated either hypoxically or aerobically. In contrast, there was a 24-fold enhancement in the differential hypoxic/aerobic response to SR 4233 in clonogenic studies. Following incubation of cells with nitrogen mustard, DNA cross-linking is observed. Bioreduction of nitromin would be expected to yield nitrogen mustard as the putative reactive metabolite. However, only DNA strand-breaks could be detected in Walker cells incubated with nitromin, suggesting that reduction of this pro-drug to nitrogen mustard was not a major activation pathway. In cells incubated under aerobic conditions, SR 4233 induces oxidative DNA damage, as indicated by the formation of 8-hydroxydeoxyguanosine, suggesting the involvement of futile redox cycling. In rats dosed with SR 4233 in vivo, significantly higher levels of 8-hydroxydeoxyguanosine could be detected in liver, compared to vehicle-dosed controls.

Comparative evaluation of combined irradiation and hyperthermia versus irradiation alone

Controvesy remains as to the treatment schedule producing better results in combined hyperthermia and X-ray therapy. Our experience concerning combined therapy of the solid tumour Walker carcinoma is reported. Male Wistar rats were submitted to treatment on the ninth day after transplantation of the tumour. Two groups of rats received either a therapeutic X-ray dose of 800 cGy by a 6-MeV linear accelerator (Mevatron, Siemens) or treatment by 432 MHz of microwaves with continuous control of tumour tissue temperature to 44 ± 1 °C for 45 min. Another group of rats was submitted to a combined treatment, with X-ray therapy preceding hyperthermia by 24 h. The last group of animals constituted the control rats. Greater tumour regression and longer survival times were obtained with the combined treatment. The gain factor for survival time was equal to 1.85 after combined treatment compared with 1.30 after X-ray therapy and 1.05 after hyperthermia. In conclusion, the results suggest that in the above schedule of combined treatment, hyperthermia applied to a solid tumour 24 h after a single dose of X-rays enhances the beneficial effect of therapy.

Augmentation of tumour delivery of macromolecular drugs with reduced bone marrow delivery by elevating blood pressure

Effects of angiotensin II (AT-II)-induced hypertension on the distribution of macromolecules to Walker carcinoma and to bone marrow of SMANCS [poly(styrene-co-maleic-acid)-neocarzinostatin conjugate] were investigated in rats. AT-II-induced hypertension from about 100 to 150 mmHg significantly increased the accumulation of the macromolecular drug SMANCS and 51Cr-labelled bovine serum albumin ([51Cr]BSA), representatives of macromolecular drugs, in tumour tissue. At 1 h after i.v. administration, intratumour concentrations of [51Cr]BSA and SMANCS were elevated by 1.2-1.8-fold. The higher drug accumulation in the tumour that was produced by the artificial hypertension was retained even 6 h after administration. This observation indicates an additive effect to that under normotensive conditions where intratumour macromolecular drug concentrations increase steadily during this period. Furthermore, distributions of these drugs in the bone marrow and the small intestine decreased during artificial hypertension to 60-80% of those in the normotensive state. Therefore, the drug concentration ratios of tumour/bone marrow and tumour/small intestine were increased by 1.8-2.4-fold. A decreased distribution of SMANCS to normal tissues under hypertensive conditions was also confirmed by the significant reduction of its toxicity e.g. leukopenia, diarrhoea, and body weight loss, even at a lethal dose. On the contrary, [3H]methylglucose showed no remarkable difference in tumour or bone marrow accumulation under this hypertensive condition. These results show the advantages of macromolecules over small molecules for AT-II-induced hypertension chemotherapy.

Differential effect of footshock stress on humoral and cellular immune responses of the rat

Recent, mostly “in vitro”, studies indicate that certain stress protocols may affect differentially immune mechanisms. In agreement with these studies, our “in vivo” results show that 4 consecutive days of intermittent footshock, in rats, depressed the primary antibody response, to a thymic dependent antigen, but enhanced the graft-versus-host response and the capacity to reject an inocula of walker carcinoma cells. Thus it is concluded that footshock stress is capable of oppositely affecting “in vivo” produced humoral and cellular immune responses.

Pyrrolizidine Alkaloids from the Seeds ofCrotalaria sessiliflora

Crotalaria sesselisora (L.) (Leguminosae) is widespread in Asia (1). The water and alcohol extracts have been used in Chinese folk medicine as antitumor agenls and showed inhibitory activity against several rodent tumors such as sarcoma 180, leukemia 615, and Walker carcinoma 256 (2). The pyrrolizidine alkaloid, monocrotaline, has been investigated as the active constituent (3). In our further research on these alkaloids, three pyrrolizidine alkaloids including monocrotaline have been isolated from the seeds of the title plant, and characterized by IR, mass, Hand C-NMR 2 dimensional spectral techniqucs (HHTCOH, COSY). Some literature data have been improved.

Protein turnover, synthesis and secretion of albumin in hepatocytes isolated from rats bearing Walker 256 carcinoma.

Hepatocytes isolated from rats bearing line A of Walker 256 carcinoma (WA) were used to study the turnover of total liver protein and the synthesis of albumin in comparison with ad libitum (AL) and pair-fed (PF) healthy controls. The rates of total protein synthesis by hepatocytes of WA animals were 40 and 90% higher than in AL and PF controls, respectively. The degradation of fast-turnover proteins was not affected by nutrition or by the tumor, whereas the degradation of slow-turnover proteins was slightly but significantly increased--about 24% higher in hepatocytes from WA rats than in PF controls. The combination of the two processes, synthesis and degradation, was in favor of an increased synthesis which explains the increase in liver protein content observed in vivo in WA rats. Dietary restriction did not affect the synthesis and secretion of albumin, whereas the tumor significantly reduced its synthesis by about 30%. The plasma concentration of albumin in WA rats dropped by about the same percentage compared with AL and PF animals.

Flow cytometric measurement of cell cycle kinetics in rat Walker‐256 carcinoma following in vivo and in vitro pulse labelling with bromodeoxyuridine

Flow cytometric measurements of total DNA content, cell cycle distribution, and bromodeoxyuridine (BrdUrd) uptake were made in rat Walker-256 carcinoma cells. After both in vivo and in vitro pulse labelling with BrdUrd, Walker-256 tumor cells were stained with propidium iodide (PI) to estimate the total DNA content and a monoclonal antibody against BrdUrd to estimate the relative amount of cells in S phase. BrdUrd-labelled single cell suspensions were harvested at different time intervals to determine the movement of these cells within the cell cycle. To increase BrdUrd uptake, fluorodeoxyuridine (FDU), a thymidine antagonist, was also applied in vivo and in vitro. The results indicated exponential growth characteristics for this tumor between days 5 and 8 after implantation. Tumor doubling times, derived from changes in tumor volume in vivo and from the increase in cell number in vitro were similar. The mean time for DNA synthesis was estimated from the relative movement of BrdUrd-labelled cells towards G2. The percent of cells labelled with BrdUrd and the DNA synthesis time were similar regardless of the mode of BrdUrd administration. This study demonstrates that BrdUrd labelling of rat Walker-256 carcinoma cells in vitro yields kinetic estimates of tumor proliferation during exponential growth similar to those with the administration of BrdUrd in the intact tumor-bearing rat.

Synergistic enhancement of the antiproliferative activity of cis-diamminedichloroplatinum(II) by the ether lipid analogue BM41440, an inhibitor of protein kinase C.

The new phospholipid analogue 3-hexadecylmercapto-2-methoxy-methyl-propyl-1-phosphocholine inhibits the phospholipid-calcium-dependent protein kinase, partially purified from Walker carcinoma cells with a Ki value of 0.56 microM. The compound inhibits the phorbol ester stimulated phosphorylation of the ribosomal protein S6 indicating that the depression of Ca2+-phospholipid-dependent protein kinase by the alkyl phospholipid also occurs in intact cells. The dose effect curve for the inhibition of cell proliferation by 3-hexadecylmercapto-2-methoxy-methyl-propyl-1-phosphocholine in Walker cells exhibits a close correlation to the dose effect curve for the depression of Ca2+-phospholipid-dependent protein kinase activity. Although alternative mechanisms cannot be excluded, the data suggest that the growth inhibitory activity of 3-hexadecylmercapto-2-methoxy-methyl-propyl-1-phosphocholine correlates with the inhibition of Ca2+-phospholipid-dependent protein kinase. The antiproliferative activity of 3-hexadecylmercapto-2-methoxy-methyl-propyl-1-phosphocholine is synergistically enhanced by cis-diamminedichloroplatinum(II).

Reduction of nitromin to nitrogen mustard: unscheduled DNA synthesis in aerobic or anaerobic rat hepatocytes, JB1, BL8 and Walker carcinoma cell lines

A novel route for the microsomal generation of nitrogen mustard from its N-oxide nitromin is demonstrated. The mustard was trapped as an adduct with diethyldithiocarbamate and estimated by capillary GLC. The enzyme responsible for this reduction could utilize either NADPH or NADH. Reduction occurred preferentially under anaerobic conditions. Purified cytochrome P450 reductase could carry out this reaction. Similar activities were seen using microsomal fractions from rat liver or liver derived BL8, JB1 or Walker 256 carcinoma cells, when these were expressed on a per mg of protein basis. Unscheduled DNA synthesis (UDS) was used as an index of activation of nitromin in these cell systems. In all instances, greater induction of UDS occurred in cells incubated with nitromin under anaerobic conditions.

A phase I and pharmacokinetic study of amphethinile

Amphethinile is a new spindle poison with a novel structure that has shown activity in the L1210, ADJ/PC6 and Walker carcinoma rodent tumours. In addition the agent appeared to have an improved therapeutic ratio compared to existing spindle poisons and is well absorbed when administered orally. The starting dose for the phase I study was 40 mg m-2 (1/10th mouse LD10) and further patients were studied at 200, 400, 800 and 1200 mg m-2, dose escalation being based on pharmacological monitoring. Significant toxic effects were seen only at 800 and 1200 mg m-2. At these doses patients experienced nausea and vomiting, light headedness during the infusion and varying degrees of lethargy following therapy. Two of six patients at 800 mg m-2 developed severe pain in the tumour bearing area 1-2 h after treatment and one experienced colicky abdominal pain. At 1200 mg m-2 two patients died within 48 h of treatment from what appeared to be vascular causes. Following these episodes the trial was discontinued. Neutropenia and alopecia occurred in two patients, one at 800 and one at 1200 mg m-2. These patients achieved the highest drug exposure in terms of area under the concentration x time curve. It was not possible to achieve an AUC consistently high enough to produce cytotoxic effects due to the occurrence of dose limiting toxicities thus amphethinile cannot at present be recommended for phase II testing by the i.v. route. The dose escalation scheme based on pharmacological monitoring resulted in a considerable saving in the duration of the trial. Further evaluation of this methodology is recommended.

Differential sensitivity of histone acetylation in nitrogen-mustard sensitive and resistant cells. Relation to drug uptake, formation and repair of DNA-interstrand cross-links

Cultivation of Ehrlich-ascites tumor cells in the presence of N-mustard leads to a selection of cells with a defective choline carrier. As N-mustard employs the choline carrier for transport, this results in reduced drug uptake and in a decrease in drug sensitivity which is specific for N-mustard. Walker carcinoma cells with a stable pleiotropic resistance to a variety of alkylating agents and adriamycin exhibit no evidence for an impaired drug transport and show the same frequency of DNA-interstrand cross-links as the sensitive parental line. Both sensitive and resistant Walker cells exhibit equal capacities for repair of N-mustard induced DNA-interstrand cross-links. The inhibition of histone acetylation by N-mustard, however, was found to be significantly lower in the resistant Walker or Ehrlich cells compared to sensitive counterparts. Although the difference between N-mustard concentrations leading to half maximal inhibition of histone acetylation in sensitive and resistant cells is considerably smaller than the difference between N-mustard doses required for half maximal inhibition of cell proliferation the data suggest that—besides DNA-DNA cross-linking—the inhibition of histone acetylation has to be considered as an important alternative mechanism responsible for the cytotoxic activity of alkylating agents. Inhibition of histone acetylation is not due an accelerated deacetylation and is predominantly expressed in chromatin fractions soluble in 0.1 M NaCl after digestion with micrococcal nuclease.

A new cytotoxic, DNA interstrand crosslinking agent, 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide, is formed from 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) by a nitroreductase enzyme in walker carcinoma cells

Walker tumour cells in vivo or in vitro are exceptionally sensitive to the monofunctional alkylating agent 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) (Cobb LM et al., Biochem Pharmacol 18: 1519–1527, 1969). CB 1954 forms DNA interstrand crosslinks in a time-dependent manner in Walker tumour cells but not in non-toxically affected Chinese hamster V79 cells [(Roberts JJ et al., Biochem Biophys Res Commun 140: 1073–1078, 1986)]. However, co-culturing Chinese hamster V79 cells with Walker cells in the presence of CB 1954 renders the hamster cells sensitive to CB 1954 and leads to the formation of interstrand crosslinks in their DNA, findings indicative of the formation by Walker cells of a diffusible toxic metabolite of CB 1954. A flavoprotein, of molecular weight 33.5 kDa as estimated by SDS-polyacrylamide gel electrophoresis, has been isolated from Walker cells and identified as a form of NAD(P)H dehydrogenase (quinone) (DT diaphorase, EC 1.6.99.2). This enzyme, in the presence of NADH or NADPH, catalyses the aerobic reduction of CB 1954 to 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide. This new compound can form interstrand crosslinks in the DNA of Chinese hamster V79 cells to which it is also highly toxic.

Tissue-specific effects of rapid tumour growth on lipid metabolism in the rat during lactation and on litter removal

1. The effect of tumour burden on lipid metabolism was examined in virgin, lactating and litter-removed rats. 2. No differences in food intake or plasma insulin concentrations were observed between control animals and those bearing the Walker-256 carcinoma (3-5% of body wt.) in any group studied. 3. In virgin tumour-bearing animals, there was a significant increase in liver mass, blood glucose and lactate, and plasma triacylglycerol; the rate of oxidation of oral [14C]lipid to 14CO2 was diminished, and parametrial white adipose tissue accumulated less [14C]lipid compared with pair-fed controls. 4. These findings were accompanied by increased accumulation of lipid in plasma and decreased white-adipose-tissue lipoprotein lipase activity. 5. In lactating animals, tumour burden had little effect on the accompanying hyperphagia or on pup weight gain; tissue lipogenesis was unaffected, as was tissue [14C]lipid accumulation, plasma [triacylglycerol] and white-adipose-tissue and mammary-gland lipoprotein lipase activity. 6. On removal (24 h) of the litter, the presence of the tumour resulted in decreased rates of lipogenesis in the carcass, liver and white and brown adipose tissue, decreased [14C]lipid accumulation in white adipose tissue, but increased accumulation in plasma and liver, increased plasma [triacylglycerol] and decreased lipoprotein lipase activity in white adipose tissue. 7. The rate of triacylglycerol/fatty acid substrate cycling was significantly decreased in white adipose tissue of virgin and litter-removed rats bearing the tumour, but not in lactating animals. 8. These results demonstrate no functional impairment of lactation, despite the presence of tumour, and the relative resistance of the lactating mammary gland to the disturbance of lipid metabolism that occurs in white adipose tissue of non-lactating rats with tumour burden.

Albumin microspheres as a drug delivery system for epirubicin: pharmaceutical, pharmacokinetic and biological aspects

The aim of the present work was to investigate the effectiveness of epirubicin-loaded microspheres to affect specific organ or tissue sites where tumors are present. Egg albumin microspheres were prepared using a heat denaturation method. The kinetics of in vitro release of the drug complied partially with first order ( r = 0.9894) as well as with diffusion model from a matrix ( r = 0.9982). The pharmacokinetics of epirubicin in serum, heart and lungs after a single i.v. dose of free epirubicin and epirubicin-loaded microspheres in rats could be described by the equation of the two-compartment open model. The pharmacokinetic parameters calculated suggest the possibility to improve the selective entrapment of epirubicin-loaded microspheres by the lungs. The aim of our efficacy studies was to evaluate intratumoral administration of epirubicin-loaded microspheres in mice with Ehrlich ascites carcinoma, as well as experimental metastases in 256 rats with Walker carcinoma. The difference between the mean survival time, respectively the final metastatic incidences of the animals from treated groups compared to controls and to the free-epirubicin group, were statistically significant. These findings suggest the possibility of organ targeting and of reducing unwanted side-effects, especially the cardiotoxicity, of the epirubicin-loaded microspheres in cancer chemotherapy.

Autonomy extends to the arterial supply of rapidly growing tumors. Studies on the feeder vessel to carcinoma in rat testis.

There has been longstanding interest in the characteristics of "feeder vessels" to tumors. To develop a convenient model for assessing the determinants of the responsiveness of the arterial blood supply to tumors, we assessed by arteriography the response of the spermatic artery to norepinephrine, vasopressin, and ergonovine after implantation of Walker carcinoma in the testis of the rat. Loss of response to norepinephrine of the spermatic artery supply to the tumor occurred by the fourth day of tumor growth in this model, along the entire length of the spermatic artery. As expected, there was sustained vasoconstriction of the contralateral spermatic artery to the normal testis in response to norepinephrine. Vasopressin and ergonovine exerted no effect, so that the specificity of the loss of response to norepinephrine remains unclear. The spermatic artery provides an accessible and convenient model for studying the blood supply to tumors.

Inhibition of cis-diamminedichloroplatinum (II)--induced renal toxicity in the rat.

The hydroxyl-containing dithiocarbamates, sodium (di(hydroxyethl)-dithiocarbamate (NaY) and sodium N-methyl, N-dithiocarboxy-D-glucamine (NaG), appear to possess definite advantages over sodium diethyldithiocarbamate (DDTC) in reducing the cis-dichlorodiammineplatinum (Cis-Pt)-induced renal damage in rats given Cis-Pt as an IV bolus of 7.5 mg/kg 1 h before the IP administration of the dithiocarbamate. Renal damage, as estimated by blood urea nitrogen (BUN) values and serum creatinine levels, was less at all times up until sacrifice in animals given NaY or NaG than in those given DDTC. An even more effective method for suppression of Cis-Pt renal toxicity is to use a combination of procedures. The most efficacious combination involves a 24-h pretreatment with DDTC or NaG plus acetazolamide and normal saline hydration 30 min before administration of Cis-Pt, followed by post-treatment with NaG. With this combination therapy renal function can be almost completely spared. Although DDTC or NaG pretreatment is highly effective when used in conjunction with NaG post-treatment, DDTC or NaG pretreatment alone has no renal sparing effect on renal function or renal platinum accumulation. In experiments in which antidotes were given 1 h after Cis-Pt and the animals were followed up for 75 days, a chronic interstitial nephritis at 75 days, suggesting a persistent cell-mediated immune response to Cis-Pt-induced renal damage, may be the basis for chronically abnormal renal function resulting from Cis-Pt. Treatment with all three dithiocarbamates, NaY, NaG, and DDTC, reduced the intensity of this cellular reaction and also reduced platinum levels in the kidneys. Although NaY and NaG are effective heavy metal chelators and renal function is spared and kidney platinum levels are substantially reduced by the dithiocarbamates, no parallel loss of antineoplastic activity by Cis-Pt on the rat Walker carcinoma was observed. Since the dithiocarbamates have no known human toxicity that would disqualify their clinical use, phase 1 clinical trials are indicated.

Alkylating antitumor agents reduce histone acetyl-transferase activity

N-Mustard depresses the acetylation of histones in Ehrlich ascites and Walker carcinoma cells. It is demonstrated that this effect is not caused by an accelerated deacetylation but is due to an inhibition of the acetyl-transferase reaction. Employing 4-sulphonatoethylthio-cyclophosphamide it is demonstrated that the alkylating agent affects predominantly the acetylation of a chromatin fraction which is soluble in 0.1 m NaCl after digestion with micrococcal nuclease. After removal of the alkylating agent, the recovery of histone acetylation is relatively slow and — in contrast to the repair of DNA cross-links — characterized by a 4-hr lag period. The reduction of histone acetylation by N-mustard is much less expressed in cells which are resistant to the drug than in the sensitive parental lines. This is in contrast to DNA-interstrand cross-links in Walker cells where both N-mustard sensitive and resistant cells inhibit the same cross-link frequency and identical repair rates. Based on these data it is concluded that the inhibition of histone acetylation may be an important part of the mechanism by which alkylating agents inhibit tumor growth.