Measurements of chromogranin B can serve as a complement to chromogranin A

OBJECTIVE: CgA has been shown to be an excellent marker for neuroendocrine tumours. However, there are two major drawbacks with CgA measurements; elevated levels are common in patients with decreased renal function and in patients on treatment with proton pump inhibitors. These problems are not seen with CgB measurements. We have recently presented the development of 13 region-specific radioimmunoassays for measurements of CgB. A region-specific assay was identified, which measured higher concentrations of CgB than the other assays and seemed to be very useful as a marker for neuroendocrine tumours. The aim of the present study was therefore to further explore the diagnostic potential of this assay in the clinical management of patients with neuroendocrine tumours. METHODS: Measurements of CgB with two methods were compared with CgA in plasma samples from patients investigated for neuroendocrine tumours (N=86), patients with decreased renal function (N=35) and patients on treatment with proton pump inhibitors (N=29). RESULTS: The diagnostic sensitivity for the new CgB assay was almost as good as that for CgA. Furthermore, with CgB measurements we could avoid the falsely elevated levels of CgA found in patients with decreased renal function and treatment with proton pump inhibitors. CONCLUSIONS: We conclude that the new CgB assay can serve as a complement to CgA measurements as an important tumour marker for neuroendocrine tumours.     

Alterations In Gastrointestinal Steady-State Kinetics Associated With the Growth of Experimental Tumours

Changes in the growth kinetics of the intestinal epithelium were observed in mice bearing the Lewis lung carcinoma and the T1699 mammary adenocarcinoma and in rats bearing the H-4-II-E₂ hepatoma. Proliferative activity in the jejunal tissue was markedly depressed with increasing tumour burden. Simultaneously, a significant reduction in total crypt cellularity occurred, followed by a reduction in villus height. While the total number of proliferative cells per crypt decreased, the relative proliferative compartment within the shrinking crypt increased. the rate of mucosal DNA synthesis remained constant during the initial cytokinetic changes, falling only after proliferative activity of the intestine was reduced to less than 50% of control levels. No general correlation could be drawn from the three tumour models studied between the level of gastrointestinal proliferation and tumour size, tumour growth rate or loss of weight by the tumour-bearing animals. However, intestinal proliferation was reduced by 50% when the tumour burden for each of the three tumours reached 6–8% of the host animal weight.     

Measurements of secretogranins II, III, V and proconvertases 1/3 and 2 in plasma from patients with neuroendocrine tumours

INTRODUCTION: Chromogranin (Cg) and secretogranin (Sg) are members of the granin family of proteins, which are expressed in neuroendocrine and nervous tissue. In recent publications we have presented generation of region-specific antibodies against CgA and CgB and also development of several region-specific radioimmunoassays for measurements of specific parts of the Cgs. In this study we describe generation of antibodies against SgII, SgIII, SgV and the proconvertases PC1/3 and PC2 and development of radioimmunoassays for measurements of these proteins. MATERIALS AND METHODS: Peptides homologous to defined parts of the secretogranin and proconvertase molecules were selected and synthesised. Antibodies were raised, radioimmunoassays were developed and circulating levels of the proteins in plasma samples from 22 patients with neuroendocrine tumours were measured in the assays. RESULTS: Increased plasma concentrations were recorded in 11, 4 and 3 of the patients with the SgII 154-165 (N-terminal secretoneurin), the SgII 172-186 (C-terminal Secretoneurin) and the SgII 225-242 assays respectively. The SgIII, SgV, PC1/3 and PC2 assays failed to detect increased concentrations in any of the patients. CONCLUSION: Increased concentrations of SgII, especially the N-terminal part of secretoneurin could be measured in plasma from patients with endocrine pancreatic tumours and in this case this assay was quite comparable to measurements of CgA and CgB. Even though secretoneurin was not as frequently increased as CgA and CgB in patients with carcinoid tumours or pheochromocytoma it may be a useful marker for endocrine pancreatic tumours.     

Distribution of Adriamycin in mice bearing mammary adenocarcinoma 16/C

The response of solid mammary adenocarcinoma 16/C to treatment with Adriamycin is highly variable and ranges from growth under treatment to complete regression. Tumour and host factors were evaluated to determine the influence of each on the response. We determined that the concentration of Adriamycin in plasma and tumour was a function of tumour size and treatment history in mice bearing mammary adenocarcinoma 16/C. The plasma concentrations following a single dose of Adriamycin (10 mg/kg) increased in proportion to tumour mass without a concurrent increase in tumour concentration. When mice bearing large tumours (greater than 1.0 g) were treated with a multidose protocol, the plasma concentrations were higher and the tumour concentrations lower following the initial dose than following subsequent doses; in tumour-free mice, prior treatment with Adriamycin did not affect the plasma level achieved after a second dose. The magnitude of the decrease in plasma and increase in tumour concentrations was a function of the initial tumour size and the treatment schedule. The increase in tumour levels represented the sum of residual Adriamycin and drug bound as a result of the dose immediately prior to analysis. At the time of the initial treatment, the Adriamycin was distributed within each tumour in proportion to vascular perfusion. The percent of the tumour mass that was well-perfused appeared to increase with repeated treatments. The results indicate that the plasma concentration of Adriamycin did not necessarily reflect the tumour exposure in the mammary adenocarcinoma 16/C model. In hosts bearing mammary adenocarcinoma 16/C--or, possibly, other tumours that produce similar effects on the host--a low initial dose of Adriamycin might modify the distribution, possibly reduce the toxicity and allow escalation of subsequent doses with increased exposure of the tumour.     

Chromogranin A correlates with norepinephrine release rate.

Chromogranin A (CgA) is an acidic protein co-released with catecholamines during exocytosis from sympathetic nerve terminals and chromaffin cells. Previous work has demonstrated that large scale perturbations in sympathetic nervous system (SNS) functioning result in corresponding changes in CgA levels in plasma. Little is known about the physiologic significance of CgA. We hypothesized that, since CgA and catecholamines are co-released from the same storage vesicles, and since CgA is not subject to reuptake or enzymatic metabolism, plasma CgA should reflect norepinephrine release from sympathetic terminals. We therefore measured venous CgA, norepinephrine levels, and norepinephrine release rate in 30 unmedicated subjects. Although the correlation of CgA with plasma norepinephrine was only modest (r = 0.37, p less than 0.05), its correlation with norepinephrine release rate was highly significant (r = 0.58, p less than 0.001). Thus, CgA may offer a novel perspective on peripheral sympathetic activity.     

Triple therapy with octreotide,galanin, and serotonin reduces the size and blood vessel density and increases apoptosis of a rat colon carcinoma

A rat colonic adenocarcinoma was implanted subcutaneously in female nude (C57BL/6JBom-nu) mice. After 7 days, the animals were divided into different groups. One group received triple therapy with octreotide, galanin, and serotonin, 10 microg/kg body weight of each, twice daily. The second group served as controls and received only saline solution. Three groups received 10 microg/kg body weight twice daily of octreotide, galanin, or serotonin. The last group consisted of controls that received only saline solution. The treatment lasted for 5 days. The tumour volume, wet weight, and relative volume density of blood vessels were significantly decreased after the triple treatment, as compared to controls. Apoptotic index was significantly increased, but the proliferation index was not affected in the group of mice that received triple therapy. There was no significant difference between controls and mice treated with octreotide, galanin, or serotonin regarding tumour volume or weight. The relative volume density of blood vessels was decreased in tumours treated with galanin, but not with octreotide or serotonin. There was no statistical difference in the proliferation index between controls and animals treated with octreotide, galanin, or serotonin, as compared with controls. Tumour necrosis and increased apoptosis may be responsible for the reduction in the volume and weight of the tumour after triple therapy. Tumour necrosis may be caused by the induction of tumour ischemia due to a reduction in tumour blood flow, which is caused by decreased incidence of tumour-feeding blood vessels, and by constriction of tumour-feeding arterioles. These results are promising and may offer treatment for colon cancer.     

Treatment with HPMA copolymer-based doxorubicin conjugate containing human immunoglobulin induces long-lasting systemic anti-tumour immunity in mice

Linkage of doxorubicin (Dox) to a water-soluble synthetic N-(2-hydroxypropyl)methacrylamide copolymer (PHPMA) eliminates most of the systemic toxicity of the free drug. In EL-4 lymphoma-bearing C57BL/6 mice, a complete regression of pre-established tumours has been achieved upon treatment with Dox–PHPMA–HuIg conjugate. The treatment was effective using a range of regimens and dosages, ranging from 62.5 to 100% cured mice treated with a single dose of 10–20 mg of Dox eq./kg, respectively. Fractionated dosages producing lower levels of the conjugate for a prolonged time period had substantial curative capacity as well. The cured mice developed anti-tumour protection as they rejected subsequently re-transplanted original tumour. The proportion of tumour-protected mice inversely reflected the effectiveness of the primary treatment. The treatment protocol leading to 50% of cured mice produced only protected mice, while no mice treated with early treatment regimen (i.e. starting on day 1 after tumour transplantation) rejected the re-transplanted tumour. Exposure of the host to the cancer cells was a prerequisite for developing protection. The anti-tumour memory was long lasting and specific against the original tumour, as the cured mice did not reject another syngeneic tumour, melanoma B16-F10. The immunity was transferable to naïve recipients in in vivo neutralization assay by spleen cells or CD8⁺ lymphocytes derived from cured animals. We propose an effective treatment strategy which eradicates tumours without harming the protective immune anti-cancer responses.     

Plasma adrenomedullin concentrations in patients with adrenal pheochromocytoma.

BACKGROUND: The hypotensive peptide adrenomedullin was first isolated in extracts of human pheochromocytoma. There is, however, no information available on the behaviour of circulating adrenomedullin or on the correlation with catecholamines in patients with pheochromocytoma. OBJECTIVES: 1) to investigate whether plasma adrenomedullin levels were changed in 10 patients with pheochromocytoma when compared to 21 healthy subjects and 16 patients with essential hypertension; 2) to determine whether or not adrenomedullin has a counter-regulatory role in catecholamine excess in pheochromocytoma or is responsible for hemodynamic modifications before and after tumour resection; 3) to determine tissue distribution of iradrenomedullin in the pheochromocytoma. METHODS: Plasma adrenomedullin and catecholamine levels were measured in all patients with pheochromocytoma before and four weeks after tumour removal. In the four patients undergoing resection of tumours, plasma levels of adrenomedullin were measured at different time-points during surgery. RESULTS: The mean plasma adrenomedullin concentrations ( SD) in patients with pheochromocytoma (37.9 +/- 6pg/ml) were significantly higher (p<0.0001) than those in normal subjects (13.7 +/- 6.1 pg/mI) and patients with essential hypertension (22.5 +/- 9.lpg/ml). Adrenomedullin levels correlated with plasma noradrenaline (r = 0.516, p = 0.0124). In all patients with pheochromocytoma, plasma adrenomedullin concentrations decreased after removal of tumours (from 37.9 +/- 6 to 10.9 +/- 4.6 pg/ml; p < 0.0001). In the four patients studied during surgery, baseline plasma adrenomedullin and noradrenaline levels were markedly elevated, and increased significantly with tumour manipulation, decreasing 24 hours after operation. Adrenal medulla cells surrounding the pheochromocytoma site stained for ir-adrenomedullin, whereas only isolated cells of pheochromocytoma stained for the peptide. CONCLUSIONS: This study demonstrates that circulating adrenomedullin is increased in pheochromocytoma, and is also correlated with plasma noradrenaline levels. Adrenomedullin may represent an additional biochemical parameter for clinical monitoring of patients with pheochromocytoma.     

The multi-site tumour transplantation model for human endometrial carcinoma: a statistical evaluation

We studied the effect of multi-site tumour transplantation on tumour growth by implanting varying numbers of EnCa 101 human endometrial tumours in athymic mice. One treatment group received a single tumour per mouse, another group received two tumours per mouse and a third group received four tumours per mouse. Tumour growth was sustained in all animals by implantation of oestradiol-17 beta pellets. We observed positive correlation between tumours within the same mouse, which implies that individual tumours are not statistically independent. The correlation is sufficiently large that failure to account for it in statistical design and analysis could result in studies with insufficient power and in spurious assertions of significance. Regression modelling of tumour growth curves showed that mean tumour volume per animal is not affected by the number of tumours growing on the animal; that is, the data are consistent with the null hypothesis that mean tumour volume is the same regardless of the number of tumours present. Our results therefore suggest that the use of multiple tumours per animal can increase the precision of experiments without loss of validity and at relatively little cost. However, correct and efficient analysis of the data so obtained requires more sophisticated techniques than those--such as fixed-effects analysis of variance and the two sample t-test--that assume independence of tumours.     

A panel of 11 region-specific radioimmunoassays for measurements of human chromogranin A

INTRODUCTION: The primary structure of human chromogranin A (CgA) not only contains 10 pairs of basic amino acids, which are potential cleavage sites for specific endogenous proteases, but also other sites in the molecule can be subjected to cleavage. Several CgA-related peptides have been identified in tissue, and many of the biological effects attributed to CgA seem to be mediated by these peptides. MATERIALS AND METHODS: Peptides homologous to defined parts of the human CgA molecule were selected and synthesised. Antibodies were raised, and 11 specific radioimmunoassays were developed. Plasma samples from 20 patients with neuroendocrine tumours were collected and measured in all assays. RESULTS: All assays measured circulating levels of CgA-derived peptides. Only four of the assays measured concentrations that correlated with that of total CgA. However, concentrations of the individual CgA-related peptides were generally lower than the concentration of total CgA. Different neuroendocrine tumours seem to process CgA differently. The ratio between a given region-specific assay and total CgA is inversely correlated to tumour activity. CONCLUSION: The assays presented allow measurements of defined regions of CgA and will thus become important tools for further studies of processing of CgA.     

Chronic infusion of norepinephrine into the VMH of normal rats induces the obese glucose-intolerant state

Increases in ventromedial hypothalamic (VMH) norepinephrine (NE) levels and/or activities have been observed in a variety of animal models of the obese insulin-resistant condition. This study examined the metabolic effects of chronic NE infusion (25 nmol/h) into the unilateral VMH of normal rats. Within 4 days, VMH NE infusion significantly increased plasma insulin (140%), glucagon (45%), leptin (300%), triglyceride (100%), abdominal fat pad weight (50%), and white adipocyte lipogenic (100%) and lipolytic (100%) activities relative to vehicle-infused rats. Furthermore, isolated islet insulin secretory response to glucose (15 mM) within 4 days of such treatment was increased over twofold (P < 0.05). Among treated animals, fat stores continued to increase over time and plateaued at approximately 2 wk (3-fold increase), remaining elevated to the end of the study (5 wk). By week 4 of treatment, NE infusion induced glucose intolerance as evidenced by a 32% increase in plasma glucose total area under the glucose tolerance test curve (P < 0.01). Whole body fat oxidation rate measured after 5 wk of infusion was significantly increased among treated animals as evidenced by a reduced respiratory quotient (0.87 +/- 0.01) relative to controls (0. 90 +/- 0.01). VMH NE infusion induced hyperphagia (30%) only during the first week and did not affect body weight over the 5-wk period. Increases in VMH NE activity that are common among obese insulin-resistant animal models can cause the development of this obese glucose-intolerant (metabolic) syndrome.     

An in vivo model of melanoma: treatment with ATP

Athymic mice, injected with A375 human melanoma cells, were treated daily with intraperitoneal injections of adenosine 5′-triphosphate (ATP). The tumour volume and animal weight were measured over the course of the experiment and the final tumour nodule weight was measured at the end of the experiment. Tumour volume decreased by nearly 50% by 7 weeks in treated mice. Weight loss in untreated animals was prevented by ATP. Histological examination of the excised tumour nodules showed necrosis in the ATP-treated tumours only. The presence of P2Y₁ and P2X₇ receptors, previously proposed as extracellular targets for melanoma treatment with ATP, were demonstrated in the excised specimens by immunohistochemistry. This paper provides further support for the use of ATP as a treatment for melanoma.     

Cancare-a herbal formulation inhibits chemically induced tumours in experimental animals

Cancer chemopreventive potential of Cancare, a multi-herbal formulation on chemically induced tumours was studied by N-nitrosodiethylamine (NDEA) induced hepatocarcinogenesis in rats and 20-methylcholanthrene (20-MC) induced sarcoma development in mice. Oral administration of Cancare was found to inhibit the liver tumour development induced by N-nitrosodiethylamine. Animals administered with NDEA had visible liver tumours by the end of 30th weeks and the liver weight was raised to 6.1 +/- 1.4 g/ 100 g body wt. None of the animals treated with Cancare (150 mg/ kg) developed any visible liver tumours by this period and the liver weight was 3.0 +/- 0.6 g/ 100 g body wt. Gamma-Glutamyl transpeptidase, a marker of hepatocellularcarcinoma, which was raised to 83.7 +/- 8. 9 U/l in serum of NDEA treated group was reduced to 35.2 +/- 6.1 U/l by simultaneous administration of Cancare. Elevated levels of serum alkaline phosphatase, glutamate pyruvate transaminase, bilirubin, liver glutathione S-transferase, glutathione and gamma-Glutamyl transpeptidase in the NDEA administered group was significantly reduced by Cancare administration. Cancare administration inhibited the sarcoma development and increased the life span of mice administered with 20-MC dose dependently. All animals in the control group developed sarcomas by 150th day and dead by 174th day after 20-MC administration. Cancare administration (30 mg and 150 mg/kg) inhibited the sarcoma development (46.7 and 60%) as well as increased the life span (53.3 and 66.7%) as estimated on 240th day after 20-MC administration. The results are indicative of the chemopreventive potential of Cancare against chemically induced neoplasmas.     

Clonidine reduces elevated cerebrospinal fluid catecholamine levels in patients with essential hypertension

Cerebrospinal fluid (CSF) catecholamines were measured in normotensive patients and in patients with mild to moderate essential hypertension. CSF-norepinephrine (NE) concentrations were 50% lower in the normotensive individuals (127 ± 28 vs. 240 ± 23 pg/m1) (P<0.01). In hypertensive patients, CSF-NE was inversely related to age (r =-0.68; P<0.01) and directly related to plasma NE (r = 0.61; P<0.05). Clonidine (450 mcg/day for 2 weeks) significantly reduced CSF-NE (?40%) in hypertensive patients. In addition, it decreased blood pressure, plasma and urinary NE. Urinary VMA was not affected by clonidine. No correlation was observed between clonidine effects on BP and on plasma or CSF catecholamines. This study indicates that patients with essential hypertension have elevated levels of CSF-NE which are reduced after treatment with clonidine. The elevation of CSF-NE suggests that central (spinal?) noradrenergic activity may be increased in patients with mild to moderate essential hypertension, and that can be reduced by treatment with clonidine.     

A single-centre experience with octreotide in the treatment of different hypersecretory syndromes in patients with functional gastroenteropancreatic neuroendocrine tumors

The aim of this research was to assess the clinical and biochemical efficacy of the octreotide in the treatment of patients with various functional gastroenteropancreatic neuroendocrine tumors (GEP-NETs). The study included 14 patients treated with octreotide for 6 months. They were diagnosed with VIPoma, glucagonoma, gastrinoma, medullary thyroid carcinoma (solitary and as a part of MEN-II syndrome), pancreatic carcinoids (solitary and as a part of multiple endocrine neoplasia type-1 syndrome-MEN-1 syndrome) and midgut carcinoids. The patients presented with Verner-Morrison, glucagonoma, Zollinger Ellison and carcinoid syndrome respectively. All had a metastatic disease at the time of diagnosis and a positive octreoscan finding. Initially elevated chromogranin A (CgA) levels were detected in 11 (78.6%) and elevated 5-hydroxyindolacetic acid (5-HIAA) levels in 8 (57.1%) patients. Symptomatic efficacy assessments were made by diarrhea reductions during treatment course, and laboratory efficacy was assessed through changes in 5-HIAA and CgA levels. Assessments were made initially and following 6 months of therapy. Median urinary 5-HIAA and the number of stools decreased significantly (p = 0.016 and p = 0.009 respectively, p < 0.05) while CgA levels had the decreasing tendency but not statistically significant (p = 0.14). There was a positive correlation between the 5-HIAA reduction and the decrease in stool number at baseline and during treatment course (p < 0.05). No correlation was observed between 5-HIAA and CgA levels and also there was no correlation between CgA reduction and symptomatic improvement. The results prove octreotide to be effective in reducing symptoms and biochemical markers associated with hypersecretory syndromes of GEP-NETs.     

NOD/SCID-GAMMA Mice Are an Ideal Strain to Assess the Efficacy of Therapeutic Agents Used in the Treatment of Myeloma Bone Disease

Animal models of multiple myeloma vary in terms of consistency of onset, degree of tumour burden and degree of myeloma bone disease. Here we describe five pre-clinical models of myeloma in NOD/SCID-GAMMA mice to specifically study the effects of therapeutic agents on myeloma bone disease. Groups of 7–8 week old female irradiated NOD/SCID-GAMMA mice were injected intravenously via the tail vein with either 1x10⁶ JJN3, U266, XG-1 or OPM-2 human myeloma cell lines or patient-derived myeloma cells. At the first signs of morbidity in each tumour group all animals were sacrificed. Tumour load was measured by histological analysis, and bone disease was assessed by micro-CT and standard histomorphometric methods. Mice injected with JJN3, U266 or OPM-2 cells showed high tumour bone marrow infiltration of the long bones with low variability, resulting in osteolytic lesions. In contrast, mice injected with XG-1 or patient-derived myeloma cells showed lower tumour bone marrow infiltration and less bone disease with high variability. Injection of JJN3 cells into NOD/SCID-GAMMA mice resulted in an aggressive, short-term model of myeloma with mice exhibiting signs of morbidity 3 weeks later. Treating these mice with zoledronic acid at the time of tumour cell injection or once tumour was established prevented JJN3-induced bone disease but did not reduce tumour burden, whereas, carfilzomib treatment given once tumour was established significantly reduced tumour burden. Injection of U266, XG-1, OPM-2 and patient-derived myeloma cells resulted in less aggressive longer-term models of myeloma with mice exhibiting signs of morbidity 8 weeks later. Treating U266-induced disease with zoledronic acid prevented the formation of osteolytic lesions and trabecular bone loss as well as reducing tumour burden whereas, carfilzomib treatment only reduced tumour burden. In summary, JJN3, U266 or OPM-2 cells injected into NOD/SCID-GAMMA mice provide robust models to study anti-myeloma therapies, particularly those targeting myeloma bone disease.     

Effect of local interleukin-2 treatment on spontaneous tumours of different immunogenic strength

Transplantable tumour lines established from spontaneous tumours of BALB/c, CBA, and DBA/2 mice displayed different immunogenic strength. This report describes tumour susceptibility to interleukin-2 (IL-2) therapy in relation to tumour immunogenicity. The following tumour lines were used: X5, X6, and X9 mammary tumours of DBA/2, BALB/c, and CBA origin respectively, X7 carcinoma of BALB/c and X18 papilloma of CBA mice. Two spontaneous tumours of long transplantation history, SL2 lymphoma (SL2) of DBA/2 and Madison lung carcinoma M109 (M109) of BALB/c origin, were used as control systems. Experimental mice were transplanted with different inocula of tumour cells at day 0; treatment with IL-2 was initiated on days 1–3 or delayed until day 10 and consisted of daily injections of low doses of 5000 or 20 000 U/mouse given five times a week for a period of 3 weeks. Treatment of SL2 (2 × 10⁴ cells injected i.p.) consisted of i.p. injections of 5000 or 20 000 U IL-2/mouse given on days 10–14 after tumour transplantation. IL-2 therapy of SL2-bearing DBA/2JIco mice resulted in a significant proportion of cures; however, no response to IL-2 treatment was achieved in SL2-bearing DBA/2CrIiw mice. BALB/c mice with the i.p. transplant of M109 responded to IL-2 treatment with 40% increase in lifespan. The low-dose IL-2 therapy of the five spontaneous tumours resulted, in general, in transient growth inhibition of the i.m. transplants of lines X5, X6, and X7 provided that IL-2 was administered locally. The therapeutic effect depended on the number of transplanted tumour cells, the best results being achieved at cell numbers close to the dose-inducing tumour growth in 50% of animals. We found that the spontaneously arising tumours responding to IL-2 treatment were all slowly growing and immunogenic (X6 and X7) or might have viral association (X5) and, as such, might express foreign antigens. The data suggest a correlation between tumour immunogenicity and the therapeutic effect. However, IL-2 can still exert some effect against tumours with negligible immunogenicity. Received: 16 July 1998 / Accepted: 5 October 1998     

Combined Micro-PET/Micro-CT Imaging of Lung Tumours in SPC-raf and SPC-myc Transgenic Mice

Introduction

SPC-raf and SPC-myc transgenic mice develop disseminated and circumscribed lung adenocarcinoma respectively, allowing for assessment of carcinogenesis and treatment strategies. The purpose of this study was to investigate the technical feasibility, the correlation of initial findings to histology and the administered radiation dose of combined micro-PET/micro-CT in these animal models.

Material and Methods

14 C57BL/6 mice (4 nontransgenic, 4 SPC-raf transgenic, 6 SPC-myc transgenic) were examined using micro-CT and ¹⁸F-Fluoro-deoxyglucose micro-PET in-vivo. Micro-PET data was corrected for random events and scatter prior to reconstruction with a 3D-FORE/2D-OSEM iterative algorithm. Rigid micro-PET/micro-CT registration was performed. Tumour-to-non-tumour ratios were calculated for different lung regions and focal lesions. Diffuse tumour growth was quantified using a semiautomated micro-CT segmentation routine reported earlier. Regional histologic tumour load was assessed using a 4-point rating scale. Gamma radiation dose was determined using thermoluminescence dosimeters.

Results

Micro-CT allowed visualisation of diffuse and circumscribed tumours in SPC-raf and SPC-myc transgenic animals along with morphology, while micro-PET provided information on metabolism, but lacked morphologic detail. Mean tumour-to-non-tumour ratio was 2.47 for circumscribed lesions. No significant correlation could be shown between histological tumour load and tumour-to-nontumour ratio for diffuse tumours in SPC-raf transgenic animals. Calculation of the expected dose based on gamma dosimetry yielded approximately 140 mGy/micro-PET examination additional to approximately 200 mGy due to micro-CT.

Conclusions

Combined micro-PET/micro-CT imaging allows for in-vivo assessment of lung tumours in SPC-raf and SPC-myc transgenic mice. The technique has potential for the evaluation of carcinogenesis and treatment strategies in circumscribed lung tumours.     

Diabetogenic response to streptozotocin varies among obese yellow and among lean agouti (BALB/c x VY)F1 hybrid mice

To test the hypothesis that the elevated insulin levels in obese neoplasia-susceptible yellow Avy/- mice might be a major factor stimulating tumor formation, it is necessary to use normoinsulinemic yellow mice. Although our attempt to obtain normoinsulinemic, euglycemic mice by streptozotocin treatment was unsuccessful, we did observe significant differences in the responsiveness to this treatment among mice of identical genotype. These differences were observed among female yellow Avy/A and agouti A/a (BALB/c x VY)F1 hybrid mice in the responses of body weight gain, plasma glucose, and plasma insulin levels to a single intraperitoneal injection of either 150 or 200 mg/kg streptozotocin (STZ) at 4 weeks of age followed by a 22-week observation period. Among animals treated with the high streptozotocin dose, 80% of the yellow mice gained almost no weight and became grossly hyperglycemic and hypoinsulinemic; however, only 55% of the agouti mice exhibited such a strong response. In the low dose group, 25% of the yellow mice responded with reduced body weight gain, decreased insulin, and elevated glucose levels whereas none of the agouti mice exhibited such responses. More pancreatic islet tissue mass was present in the untreated yellow control mice than among the comparable agouti mice by the end of the study. In both streptozotocin dose groups and in both genotypes, islet tissue mass was reduced to a much greater extent in the more responsive mice than in the less responsive mice. There appeared to be no correlation between islet tissue mass and insulin level. The phenotypic variation in responsiveness to an exogenous agent among test animals of a single inbred or F1 hybrid genotype reported here is not unique to this F1 hybrid since it is seen in most chronic bioassays when relatively low levels of agent are used.     

Effects of omeprazole on cell kinetics of carcinogen-induced colon tumours in rats

This study was designed to evaluate the effects of hypergastrinaemia induced via suppression of gastric acid by omeprazole on carcinogen-induced colon cancer in rats. The carcinogen methylazoxymethanol (MAM), 30 mg/kg, was administered intraperitoneally at 6-weekly intervals to Sprague-Dawley rats. Four weeks after the last MAM injection, the first daily dose of omeprazole, 40 mg/kg, was given by gastric gavage to one group of rats, and the rest were given buffered methylcellulose vehicle. After 10 weeks of daily omeprazole or vehicle, the rats were anaesthetized with ether, blood samples obtained, and animals sacrificed. Gastrin levels in serum from omeprazole-treated rats were elevated nearly six-fold. DNA and RNA levels in gastric mucosa were unchanged by omeprazole, but protein content was somewhat reduced. No biochemical or histological changes related to omeprazole treatment were observed in normal colon. The number of tumours, tumour volumes, and total tumour burden were not significantly different in colons of vehicle- or omeprazole-treated rats. Analysis by flow cytometry revealed that the S phase fraction was lower in tumour cells from omeprazole-treated animals; and that the frequency of DNA aneuploidy was also reduced. The results indicate that while omeprazole-induced suppression of stomach acid in rats elevates levels of gastrin in serum, it does not substantially alter the biochemical or cellular characteristics of carcinogen-induced colon tumours.