RES-macrophage stimulation and liver tumour growth in rats; evaluation with dynamic liver RES scintigraphy

Dynamic liver RES scintigraphy with Nanocoll (99Tcm albumin colloid - 50 nm diameter) assessing RES-macrophage phagocytic function was performed in 40 control, 73 RES-stimulated non-tumour-bearing and 59 tumour-bearing Wistar/FU rats in vivo. Tumour-bearing rats were inoculated with 10(6) x 1.0 cells of a syngeneic nitrosoguanidine-induced colonic carcinoma in the liver. Twenty-eight of these rats had been treated one day previously with Zymosan (3 mg x 100 g-1 i v) as a RES stimulant. The clearance/uptake rate (k) of Nanocoll was calculated from dynamic liver images by the slope in the plot 1n [1 - U(t)/U] versus t where t is time and U liver uptake. k-value in control animals was 0.45 +/- 0.01.10(-2) x s-1 Zymosan injection in non-tumour-bearing rats caused stati-stically significant higher clearance/uptake rate on day 1, through 8 after treatment compared to that of controls. On day 8 k-value was 0.64 +/- 0.04. In tumour-bearing rats the uptake rate (k) was on day 8 0.66 +/- 0.03, while in RES-stimulated tumour-bearing rats it was 0.64 +/- 0.03. Survival was 22 +/- 1 days in tumour bearing rats and 37 +/- 4 days in RES stimulated tumour-bearing rats. The average tumour volume after one week was 132 +/- 24 mm3 in non-stimulated rats and 20 +/- 5 mm3 in RES stimulated rats. There was a negative correlation between uptake rate and tumour volume and a positive correlation between uptake rate and survival on day 8 in non-stimulated tumour-bearing rats. Dynamic liver RES scintigraphy with small size 99Tcm albumin colloid (Nanocoll) can be used to measure RES phagocytic function and the effect of liver tumour growth on RES.     

Radiohalogenation of a monoclonal antibody using an N-succinimidyl 3-(tri-n-butylstannyl)benzoate intermediate

N-Succinimidyl 3-(tri-n-butylstannyl)benzoate (ATE) was elevated for its utility in the radiohalogenation of monoclonal antibodies. The F(ab')2 fragment of monoclonal antibody OC 125 was labeled with 125I using the ATE reagent and with 131I using a conventional electrophilic iodination method (Iodogen). N-Succinimidyl 3-[125I]iodobenzoate was synthesized from ATE in greater than 90% yield and purified using a disposable silica gel cartridge. About 60-65% of the radioiodinated product was coupled to the F(ab')2 fragment after a 30-min reaction. Two procedures were investigated, one involving exposure of antibody to 35 nmol of ATE and the other to 240 nmol of ATE. Using Scatchard analyses, affinity constants for binding to CA 125 antigen for OC 125 F(ab')2 labeled using the low ATE, Iodogen, and high ATE procedures were determined to be (5.2 +/- 1.0) x 10(10), (2.5 +/- 0.9) x 10(10), and (4.2 +/- 2.4) x 10(9) M-1, respectively. Paired-label studies in athymic mice bearing OVCAR-3 tumors treated with injections of antibody labeled via both ATE and Iodogen demonstrated that use of the ATE method (a) reduced thyroid uptake to less than 0.1% of the injected dose, more than 100 times less than that observed with Iodogen; (b) resulted in more rapid clearance of activity from normal tissues; and (c) with the low ATE preparations, increased the uptake of radioactivity in tumor from 27 to 49%. At 96 h, tumor:tissue ratios were generally at least 4-fold higher when antibody was labeled via ATE. These results suggest that the ATE method may be a valuable approach for the radiohalogenation of antibodies.     

Differing contribution of thiopurine methyltransferase to mercaptopurine versus thioguanine effects in human leukemic cells

Thioguanine and mercaptopurine are prodrugs requiring conversion into thiopurine nucleotides to exert cytotoxicity. Thiopurine S-methyltransferase (TPMT), an enzyme subject to genetic polymorphism, catabolizes thiopurines into inactive methylated bases, but also produces methylthioguanine nucleotides and methylmercaptopurine nucleotides from thioguanine and mercaptopurine nucleotides, respectively. To study the effect of TPMT on activation versus inactivation of mercaptopurine and thioguanine, we used a retroviral gene transfer technique to develop human CCRF-CEM cell lines that did (TPMT+) and did not (MOCK) overexpress TPMT. After transduction, TPMT activities were 14-fold higher in the TPMT+ versus the MOCK cell lines (P < 0.001). TPMT+ cells were less sensitive to thioguanine than MOCK cells (IC(50) = 1.10+/- 0.12 microM versus 0.55 +/- 0.19 microM; P = 0.02); in contrast, TPMT+ cells were more sensitive to mercaptopurine than MOCK cells (IC(50) = 0.52 +/- 0.20 microM versus 1.50 +/- 0.23 microM; P < 0.01). The lower sensitivity of TPMT+ versus MOCK cells to thioguanine was associated with lower thioguanine nucleotide concentrations (917 +/- 282 versus 1515 +/- 183 pmol/5 x 10(6) cells; P = 0.01), higher methylthioguanine nucleotide concentrations (252 +/- 34 versus 27 +/- 10 pmol/5 x 10(6) cells; P = 0.01), less inhibition of de novo purine synthesis (13 versus 95%; P < 0.01), and lower deoxythioguanosine incorporation into DNA (2.0 +/- 0.6% versus 7.2 +/- 2.0%; P < 0.001). The higher sensitivity of TPMT+ cells to mercaptopurine was associated with higher concentrations of methylmercaptopurine nucleotide (2601 +/- 1055 versus 174 +/- 77 pmol/5 x 10(6) cells; P = 0.01) and greater inhibition of de novo purine synthesis (>99% versus 74%; P < 0.01) compared with MOCK cells. We conclude that methylation of mercaptopurine contributes to the antiproliferative properties of the drug, probably through inhibition of de novo purine synthesis by methylmercaptopurine nucleotides, whereas thioguanine is inactivated primarily by TPMT.     

bcl-2、p53在皮肤肿瘤中的表达及意义

目的探讨bcl-2、p53在几种皮肤肿瘤中的表达及意义。方法采用流式细胞术(FCM)和免疫荧光技术对皮肤鳞状细胞癌(SCC)、恶性黑色素瘤(MM)、基底细胞癌(BCC)、色素痣(PN)bcl-2、p53蛋白的表达进行定量分析,以荧光指数(FI)作为定量表达指标。结果鳞状细胞癌、基底细胞癌的bcl-2、p53基因蛋白的FI值均显著性高于正常对照(P<0.05),基底细胞癌的bcl-2基因的FI值显著性高于鳞状细胞癌(P<0.05),而二者的p53基因蛋白的FI值无显著性差异(P>0.05);恶性黑色素瘤、色素痣的bcl-2、p53基因蛋白的FI值均显著性高于正常对照(P<0.05),恶性黑色素瘤的p53基因的FI值显著性高于色素痣(P<0.05),而二者的bcl-2基因蛋白的FI值无显著性差异(P>0.05)。结论鳞状细胞癌、恶性黑色素瘤、基底细胞癌、色素痣中均有bcl-2的表达,基底细胞癌bcl-2表达显著高于鳞状细胞癌,说明基底细胞癌的发生发展可能与细胞凋亡受抑密切相关;p53在恶性黑色素瘤的表达高于色素痣,说明p53为黑色素瘤的恶性标志,检测p53表达可以作为鉴别皮肤黑色素瘤恶性病变的辅助手段。     

High incidence of second basal cell skin cancers

We considered the risk of second basal cell cancers (BCC) of the skin using a population-based series of 1,868 BCC collected between 1976 and 1985 in the Swiss Cantons of Vaud and Neuchatel, and followed-up to the end of 2003. Overall, 507 second BCC were observed versus 59.98 expected, corresponding to a standardized incidence ratio (SIR) of 8.45 (95% CI: 7.73-9.22). The SIRs were similar in men and women in subsequent calendar periods, but tended to decline with advancing age at diagnosis of first BCC, from 13.98 below age 50 to 7.13 at age 70 or over. Consequently, the rate of first BCC increased to approximately 30-fold between 7/100,000 at age 30-39 and 200/100,000 at age 70-79, but the rate of second BCC increased only about 3-fold between 31/1,000 at age 30-39 and 110/1,000 at age 70-79. The cumulative risk of second BCC was 11% at 5 years, 21% at 10 years and 40% at 20 years. This study indicates that the relative (but not the absolute) risk of second BCC is greater at younger age and declines with advancing age, and is therefore compatible with an excess baseline risk in a population of susceptible individuals.     

Tumor necrosis factor-alpha, but not lymphotoxin, stimulates growth of tumor cells in hairy cell leukemia

We investigated the effect of recombinant tumor necrosis factor-alpha (rTNF-alpha) and recombinant lymphotoxin (rLT) in the growth modulation of purified hairy cell leukemia (HCL) cells. In response to rTNF-alpha, HCL cells from five of eight patients showed a 3 to 23-fold thymidine incorporation above their unstimulated controls. The effect was time and dose dependent with a maximum between 10 and 25 ng/ml rTNF-alpha after 120-hr incubation. rLT (1-50 ng/ml), however, could not enhance DNA synthesis in six of six cases. Cell number of rTNF-alpha stimulated cells ranged from 2-3 x 10(6)/ml from days 0-50 whereas cell number of unstimulated controls decreased from 3 x 10(6)/ml at day 0 to 0.01-0.02 x 10(6)/ml after 50 days in culture. rTNF-alpha induced proliferation could be suppressed in all HCL cell populations by 0.3 ng/ml recombinant interferon alpha (100 U/ml rIFN-alpha). TNF binding studies in two patients revealed that both TNF-sensitive HCL cells (1,990 +/- 148 receptors/cell) as well as TNF-insensitive HCL cells (1,261 +/- 101 receptors/cell) express specific receptors for TNF-alpha. These data show that rTNF-alpha and rLT have different effects on the growth of HCL cells. In addition there is a subgroup of patients who show no response to rLT or rTNF-alpha.     

The rate of increase in the numbers of primary sporadic basal cell carcinomas during follow up is associated with age at first presentation

Basal cell carcinoma (BCC) patients demonstrate marked variation in tumour numbers and site. Previous studies also show an association between age at first BCC presentation and development of BCC on the trunk. In this study we have investigated the association between age at first presentation and the rate of development of truncal and non-truncal tumours in 747 patients with BCC. We used negative binomial regression analysis to show that increasing age at first presentation was associated with an increased rate of BCC development (rate ratio 1.01/year, 95% CI 1.01-1.02, P < 0.001). In particular, development of tumours was greater in cases aged 60.0-69.9, 70.0-79.9 and 80.0-89.9 years than in those 40.0-49.9 years (P = 0.05, 0.01 and 0.039, respectively). While few cases aged over 70 years of age first present with a truncal BCC, the numbers of BCC/year were greater than in those with a head/neck BCC. The data suggest different genetic factors mediate the appearance of BCC in patients of different ages particularly those aged above and below 60 years.     

Inhibition of cis-diamminedichloroplatinum secretion by the human kidney with probenecid

The renal handling of cis-diamminedichloroplatinum (CP) was investigated by measuring the renal clearance of creatinine, inulin, and free platinum in ten cancer patients. Free platinum clearances exceeded the glomerular filtration rate in all time periods. For example, at 1 to 2 hr, the mean clearance of free platinum was 224 +/- 32 (S.E.) ml/min compared to a mean creatinine clearance of 94 +/- 15 ml/min or a mean inulin clearance of 94 +/- 17 ml/min (p less than 0.01), indicating secretion of CP or a metabolite. Seven additional cancer patients were treated with probenecid prior to CP. Fractional platinum clearances, calculated as a ratio of free platinum clearance to creatinine clearance, were reduced significantly in the probenecid-treated group (158 +/- 17%) compared to controls (270 +/- 57%) (p less than 0.03). Fractional platinum clearances, calculated as a ratio of free platinum clearance to inulin clearance, were also significantly reduced in the treated group (154 +/- 14%) compared to controls (271 +/- 47%) (p = 0.04). These results suggest that cisplatin is secreted by the human kidney, and this can be inhibited by probenecid. Such maneuvers may be helpful in improving the therapeutic index of this important chemotherapeutic agent.     

Tumor-infiltrating lymphocytes and prognosis of hepatocellular carcinoma.

Tumor-infiltrating lymphocytes (TIL) were isolated from 17 human hepatocellular carcinomas (HCC). The proliferation of TIL cultured with recombinant interleukin-2 (rIL-2) was evaluated. We also examined prognosis in relation to TIL. Successful expansion of TIL was achieved in 16 of the 17 lesions. In 10 of the 16, TIL increased more than 100-fold. Cytotoxicity to the allogeneic HCC cell line, HC-4, was demonstrated in all 13 TIL cultures tested. Maximum cytotoxicity was noted two to four weeks after culture. No statistical difference was observed either with respect to prognosis based upon growth rate or the cytotoxicity demonstrated in vitro. The initial number of TIL per unit weight of tumor was, however, significantly greater in the group for which the prognosis was good (19.0 +/- 5.1 x 10(6) vs. 5.6 +/- 1.6 x 10(6), P < 0.05). It would appear that greater lymphocytic tumor infiltration is a prognostic marker.     

Tumor necrosis factor-alpha (TNF alpha) enhances cisplatin cytotoxicity to ovarian carcinoma xenografts

Antitumor effect was compared between administration schedule of once a day dosing of cisplatin (DDP) for 2 consecutive days (q40d) with or without TNF alpha. In two controls, TNF alpha or dilutor alone was administered. Against the ovarian carcinoma 2008 cells, DDP given at dose level of daily x 2 (3.5 mg/kg/day q40d) combined with TNF alpha (50 mu g/kg/day) schedule showed 6.08-fold tumor growth delay (TGD) (17.10+/-1.65; P<0.01), produced 3.17-fold greater cell kill [ratio of tumor volume of treated and control groups (T:C)=0.148; P<0.01] and resulted in longer survival [median survival (MS)=166; P<0.01] than DDP alone. These are superior to even high dose DDP (7.0 mg/kg/day x 2d) without TNF alpha administration schedule showing TGD=11.38 days T:C=0.271 and MS=97 days. High dose DDP (7.0 mg/kg/day q40d) with TNF alpha showed further DDP antitumor potency (TGD=29.74+/-2.08, P<0.01), however, this schedule showed only 2.56-fold TGD extension and no improvement was found in survival because of its severe toxicity. TNF alpha did not alter DDP induced systemic toxicity. These data indicate that optimal antitumor activity, tolerance and survival improvement occured on a schedule of low dose DDP combined with TNF alpha, and this has prompted the clinical evaluation of this administration schedule.     

Vascular permeability in a human tumour xenograft: molecular charge dependence

Molecular charge is one of the main determinants of transvascular transport. There are, however, no data available on the effect of molecular charge on microvascular permeability of macromolecules in solid tumours. To this end, we measured tumour microvascular permeability to different proteins having similar size but different charge. Measurements were performed in the human colon adenocarcinoma LS174T transplanted in transparent dorsal skinfold chambers in severe combined immunodeficient (SCID) mice. Bovine serum albumin (BSA) and IgG were fluorescently labelled and were either cationized by conjugation with hexamethylenediamine or anionized by succinylation. The molecules were injected i.v. and the fluorescence in tumour tissue was quantified by intravital fluorescence microscopy. The fluorescence intensity and pharmacokinetic data were used to calculate the microvascular permeability. We found that tumour vascular permeability of cationized BSA (pI-range: 8.6-9.1) and IgG (pI: 8.6-9.3) was more than two-fold higher (4.25 and 4.65x10(-7) cm s(-1)) than that of the anionized BSA (pI approximately 2.0) and IgG (pI: 3.0-3.9; 1.11 and 1.93x10(-7) cm s(-1), respectively). Our results indicate that positively charged molecules extravasate faster in solid tumours compared to the similar-sized compounds with neutral or negative charges. However, the plasma clearance of cationic molecules was approximately 2x faster than that of anionic ones, indicating that the modification of proteins enhances drug delivery to normal organs as well. Therefore, caution should be exercised when such a strategy is used to improve drug and gene delivery to solid tumours.     

正常子宫颈和宫颈癌的弥散加权成像特点

背景与目的:磁共振成像(magnetic resonance imaging,MRI)是宫颈癌诊断和分期的重要检查方法.本研究对正常子宫颈和宫颈癌组织的弥散加权成像(diffusion-weighted imaging,DWI)特点进行分析,并探讨DWI在宫颈癌诊断以及放疗后疗效监测方面的价值.方法:对16例非宫颈肿瘤女性的子宫颈和20例宫颈癌患者的子宫颈进行常规MRI扫描和横断面DWI(b=800 s/mm2),比较正常宫颈和宫颈癌病灶的表观弥散系数(apparent diffusion coefficient,ADC)值.比较未行手术的7例宫颈癌患者放疗前后宫颈的ADC值.结果:正常子宫颈在DWI图上呈三层结构,其平均ADC值[(1.71±0.14)×10-3 mm2/s)]显著高于宫颈癌的ADC值[(0.97±0.13)×10-3 mm2/s)](P＜0.01).放疗后子宫颈的ADC值[(1.49±1.40)×10-3 mm2/s]较放疗前[(1.02±0.06)×10-3 mm2/s]升高,但仍低于正常子宫颈.结论:DWI能够区分正常子宫颈和宫颈癌组织,可用于宫颈癌治疗前侵犯范围的评价,并可显示放疗后宫颈组织的改变.     

IL-12转染对人卵巢癌SKOV3细胞增殖的影响及其机制

背景与目的:卵巢恶性肿瘤发病率、复发率、转移率均较高,有研究表明IL-12具有明显的抗肿瘤作用.本研究探讨白细胞介素-12(interleukin-12,IL-12)对人卵巢癌SKOV3细胞生长抑制作用机制及其对裸鼠皮下种植瘤成瘤能力的影响.方法:应用脂质体转染技术将含有小鼠IL-12全长基因的质粒转染到SKOV3细胞(SKOV3/IL-12组),同时以空白质粒载体转染SKOV3细胞(SKOV3/neo组)和未转染SKOV3细胞作为对照.ELISA法检测3组细胞上清中IL-12表达.MTT法检测3组SKOV3细胞的抑制率和细胞倍增时间.建立裸鼠皮下卵巢癌种植模型,分别接种SKOV3/IL-12、SKOV3/neo、SKOV3细胞,观察肿瘤生长情况.ELISA法检测裸鼠血清中IL-12及γ-干扰素(gamma interferon,IFN-γ)的表达,免疫组化法检测裸鼠种植瘤中血管内皮生长因子(vascular endothelial growth factor,VEGF)、微血管密度(microvessel density,MVD)及干扰素诱生蛋白-10(IFN-gamma-inducible protein 10,IP-10)的表达.结果:转染48、60 h后细胞上清中IL-12蛋白表达量,SKOV3/IL-12组[(473.0±38.0)pg/ml、(522.0±32.0)pg/ml]高于SKOV3/neo组[(16.0±1.3)pg/ml、(18.0±1.6)pg/ml]及SKOV3组[(16.0±1.2)pg/ml、(17.0±1.4)pg/ml](P＜0.01).SKOV3/IL-12组细胞增殖抑制率为63.7%,与对照组比较差异有统计学意义(P＜0.01),SKVO3/IL-12组细胞倍增时间(45.8±2.7)h明显长于SKOV3/neo细胞组(27.6±2.2)h和SKOV3组(28.2±2.1)h(P＜0.01).裸鼠皮下卵巢癌种植模型中,SKOV3/IL-12组裸鼠成瘤率(5/8)低于对照组(8/8)(P＜0.01);平均成瘤时间[(15.0±5.0)天]长于SKOV3/neo组[(7.9±3.2)天]和SKOV3组[(7.8±2.4)天](P＜0.01);SKOV3/IL-12组种植瘤体积、重量和裸鼠体重低于SKOV3/neo组和SKOV3组(P＜0.01),种植瘤生长速度缓慢,抑瘤率达61.3%.SKOV3/IL-12组裸鼠血清中IL-12、IFN-γ表达量高于SKOV3/neo组和SKOV3组(P＜0.01).SKOV3/IL-12组裸鼠种植瘤中IP-10阳性率(100%)高于对照组(P＜0.01),VEGF阳性率(62.5%)及MVD低于对照组(P＜0.01).结论:转染IL-12能有效地抑制SKOV3细胞生长;转染IL-12的SKOV3/IL-12细胞在裸鼠皮下的成瘤能力降低;IL-12可通过诱导IFN-γ诱生IP-10抑制肿瘤血管形成而实现其抗卵巢癌的作用.     

A Phase 1 study of high doses of aminopterin with leucovorin rescue in patients with advanced metastatic tumors.

We have conducted a Phase 1 study of aminopterin (AMT) with leucovorin (LV) in 17 patients. AMT was administered by bolus injection every 7 to 14 days in dosages from 25 to 425 mg/sq m. LV rescue was instituted at 24 hr and continued for 48 to 72 hr. At dosages above 50 mg/sq m, we observed nephrotoxicity defined as greater than or equal to a 25% increase in serum creatinine 24 hr after AMT administration, but its incidence was not strictly dose related. Urinary alkalinization and volume expansion appeared to reduce the incidence of nephrotoxicity. Nephrotoxic drug courses were associated with 24-hr plasma AMT levels [3.6 +/- 2.0 (S.D.) X 10(-6) M] which were significantly higher than nonnephrotoxic courses (1.6 +/- 1.0 x 10(-6) M) (p less than 0.05). In nonnephrotoxic courses, serum elimination pharmacokinetics appeared to be biphasic with a t1/2 alpha of 1.08 +/- 0.01 hr and t1/2 beta of 12.31 +/- 0.06 hr. Systemic toxicity (myelosuppression and mucositis) could be prevented in patients with impaired AMT clearance by the administration of LV at an increased dose rate. In several courses, systemic toxicity occurred in spite of apparently normal plasma clearance, suggesting that 24-hr plasma levels may not accurately reflect intracellular drug effects. Cytokinetic studies on bone marrow aspirates allowed determination of the rescue effect of LV and may prove useful in predicting marrow protection.     

Cyclooxygenase-2 is up-regulated in lung parenchyma of chronic obstructive pulmonary disease and down-regulated in idiopathic pulmonary fibrosis

BACKGROUND AND AIM OF THE WORK: In vitro studies have suggested that fibroblasts from idiopathic pulmonary fibrosis (IPF) may have an impaired induction of cyclooxygenase (Cox)-2. We have investigated Cox-1 and Cox-2 expression in lung tissue from IPF. METHODS: Cox-1 and Cox-2 expression were determined using RT-competitive PCR and immunohistochemistry in pulmonary biopsies from IPF (n = 22), chronic obstructive pulmonary disease (COPD) (n = 13), and lung tissue from subjects undergoing pleurodesis for spontaneous pneumothorax (control group, n = 17). RESULTS: Immunohistochemical analysis showed that the score of Cox-2 positive cells was higher in COPD (1 +/- 0) with respect to fibrosis (0.37 +/- 0.1, p < 0.05) and controls (0.57 +/- 0.2). There were no differences between fibrosis and controls in Cox-2 positive cells. The expression of Cox-2 mRNA was significantly higher in COPD (3.26 +/- 0.72 x 10(6) molecules cDNA/microg total RNA) in comparison to IPF (0.57 +/- 0.17) and controls (0.54 +/- 0.16) (p < 0.001). After IL-1beta stimulation (1-10 ng/ml) Cox-2 mRNA basal expression increased significantly in controls (from 35 +/- 12 to 94 +/- 4 x10(6) molecules cDNA/microg total RNA, p < 0.01) and in COPD (from 38 +/- 8 to 92 +/- 3, p < 0.01). In contrast, no significant changes in Cox-2 mRNA expression were found in IPF (from 30 +/- 12 to 43 +/- 16). CONCLUSIONS: Our results suggest that differences in Cox-2 expression may play a role in the regulation of inflammatory responses in lung diseases. Excessive activity is associated with the development of chronic obstructive lung disease, while a limited activation following pro-inflammatory stimulation might contribute to fibrogenic responses.     

Chronic alcohol consumption stimulates VEGF expression, tumor angiogenesis and progression of melanoma in mice

The mechanisms of alcohol-induced cancer in humans are unclear. We used the immunocompetent mice implanted with B16F10 cells to evaluate the effects of physiologically relevant EtOH intake on tumor growth and angiogenesis of melanoma. Six-wk-old male mice (C57BL/6J) were given 1% EtOH in drinking water for 12-hrs during the night which was then replaced with regular water during the remaining 12-hrs each day for 4 wks (n = 10). The control mice received regular drinking water only. In the second week, all mice were inoculated subcutaneously on the right proximal dorsal with 5 x 10(5) B16F10 cells. In the end, the tumors were isolated for measuring tumor size, average microvascular density (AMVD) using CD31 immunohistochemistry, and the expression of VEGF and its receptor (Flt-1) using Northern blot, ELISA, and immunohistochemistry. EtOH intake caused a 2.16-fold increase in tumor weight over the control (4.81 +/- 0.39 vs. 2.23 +/- 0.48 g; n = 10; p = 0.003), a 2.02-fold increase in AMVD (60.63 +/- 5.56 vs. 30.01 +/- 7.41/mm(2); p = 0.0014), and a significant increase in VEGF mRNA and protein expression plus Flt-1 protein levels in melanoma compared to the control group (p < 0.01). These results suggest that progression of melanoma growth and angiogenesis may be mediated by upregulation of VEGF and Flt-1, especially under the influence of EtOH.     

Comparison of the mutagenic potency of 1,3-butadiene at the hprt locus of T-lymphocytes following inhalation exposure of female B6C3F1 mice and F344 rats

1,3-Butadiene (BD) is an indirect alkylating agent that has greater cancer potency in the mouse than in the rat. The purpose of the present study was to compare the mutagenic potency of BD at the hprt locus of T- lymphocytes of exposed mice and rats and to determine whether mutations induced in this marker gene can be used as a quantitative indicator for species differences in susceptibility to cancer. To this end, experiments were conducted to define the effects of exposure duration and the time elapsed after exposures on the frequency of hprt mutations (Mf) in T-cells from female B6C3F1 mice and F344 rats of similar age (4- 5 weeks) when exposed to BD by inhalation. The accumulation of hprt mutations in T-cells from thymus was assessed in animals necropsied 2 weeks after exposure to 0 or 1250 ppm BD for 1 or 2 weeks, while the time course for the appearance of hprt mutant T-cells (i.e., the phenotypic expression and cell migration) in thymus and spleen was evaluated in animals necropsied at weekly/biweekly intervals up to 10 weeks after exposure for 2 weeks. At necropsy, T-cells were isolated from thymus and spleen and cultured in the presence of IL-2, concanavalin A, and 6-thioguanine (Walker and Skopek, Mutat. Res., 288, 151-162, 1993). BD exposures of 1 and 2 weeks led to mutagenic effects in mouse thymus, with the average Mfs being 3- and 5-fold greater than background values, respectively. In rat thymus, there was only a 1.7- fold increase in Mfs after 2 weeks of BD exposure. In the mutant expression experiment, hprt Mfs in thymus and spleen of both species increased for several weeks post-exposure and then declined. Hprt Mfs in thymus reached maximum levels at 2 weeks post-exposure in mice (Mfs = 11.3 +/- 2.4 x 10(-6)) and at 3 weeks post-exposure in rats (4.9 +/- 1.2 x 10(-6)), while hprt Mfs in spleen reached peak levels at 5 weeks post-exposure in mice (19.7 +/- 1.9 x 10(-6)) and 4 weeks post-exposure in rats (10.1 +/- 1.8 x 10(-6)). Background Mfs for mouse and rat thymus and spleen ranged from 1.6 +/- 0.3 x 10(-6) to 3.0 +/- 1.1 x 10(- 6). Statistical analyses of the hprt Mf data for spleen demonstrated that, under these exposure conditions, the mutagenic potency of BD (represented by the difference in the areas under the phenotypic expression curves of treated versus control animals) was 5-fold greater in mice than in rats. The magnitude of the species differences in mutagenic potency, observed after 2 weeks of BD exposure, resembles the species differences in metabolism more closely than the species differences in cancer potency.      

Self-diffusion of water in multicellular spheroids measured by magnetic resonance microimaging.

Nuclear magnetic resonance microimaging measurements of the self-diffusion coefficient of water in large (greater than 2 mm) EMT-6 multicellular spheroids were performed in order to elucidate diffusion mechanisms in tumors. Pulsed gradient spin echo-imaging methods were developed for measuring diffusion in an intravoxel multicompartment system. The self-diffusion coefficient (at 22 degrees C) for water in the medium (Dm) consisted of only a single diffusion compartment [Dm = 1.99 +/- 0.03 (SE) x 10(-5) cm2/s]. Similarly, the spheroid necrotic center showed a single water diffusion compartment with a self-diffusion coefficient (Dc) significantly lower than that of the medium (Dc = 1.54 +/- 0.05 x 10(-5) cm2/s). The spheroid viable rim region showed two distinct compartments of approximately equal volume, one with a large diffusion coefficient (1.70 +/- 0.12 x 10(-5) cm2/s) and a second with a significantly smaller diffusion coefficient (0.25 +/- 0.01 x 10(-5) cm2/s). We propose that these two experimentally distinguishable compartments correspond to the extra- and intracellular regions, respectively, of the viable rim of the spheroid. Although the diffusion coefficients were significantly different in the medium, the necrotic center, and the viable rim, the activation energy for diffusion was the same in the three regions (0.20 eV). Studies of perfused spheroids at 37 degrees C show the same dependence of the diffusion coefficients on the diffusion filter as observed for unperfused spheroids at 22 degrees C. These results demonstrate the ability of nuclear magnetic resonance microimaging to investigate diffusion at the cellular level, which will lead to a better understanding of microenvironmental regulation in tumors.     

MC1R variants associated susceptibility to basal cell carcinoma of skin: interaction with host factors and XRCC3 polymorphism

The variants within the human melanocortin 1 receptor (MC1R) gene are associated with an increased risk of different skin cancers. In this study, we genotyped by direct sequencing, 529 cases of basal cell carcinoma of the skin (BCC) and 533 healthy controls for polymorphisms in the entire MC1R gene. In addition to 10 common polymorphisms, we detected 23 rare variants in the gene. The presence of any nonsynonymous MC1R variant was associated with an increased risk in the carriers (odds ratio OR 1.66, 95% confidence interval CI 1.28-2.14) corresponding to a population attributable fraction of about 27%. The odds ratio for the risk in the carriers of 2 MC1R variants was 2.69 (95% CI 1.77-4.08). The risk of BCC in the carriers of MC1R variants with fair complexion was almost twice as much as in the corresponding noncarriers. The carriers of the R163Q variant with a medium skin complexion were at a 3-fold higher risk than the noncarrier counterparts. The interaction, of effect on the BCC risk, between the MC1R variants and types of skin response to sun exposure was greater than multiplicative. We also observed a multiplicative interaction of risk due to the MC1R variants and the common allele (high risk) of the T241M polymorphism in the XRCC3 gene. Our data confirmed the status of the nonsynonymous MC1R variants as independent genetic risk factors for BCC. However, the mechanism through which the variants influence the risk likely involves complex interactions with other genetic and host risk factors.     

Adjustment of creatinine clearance improves accuracy of Calvert's formula for carboplatin dosing.

Carboplatin clearance depends on the glomerular filtration rate (GFR), and Calvert''s formula is frequently used to achieve a target area under the time vs concentration curve (mg ml(-1) min). Creatinine clearance is a substitute for GFR when creatinine values are determined by the Jaffé method, which is being replaced by the enzymatic method. When the enzymatic method is used, the corresponding creatinine clearance theoretically exceeds GFR, and the use of creatinine clearance as GFR in Calvert''s formula results, accordingly, in overdosing of carboplatin. In this study, we have established a model for adjusting the creatinine clearance to offset this bias based on a relationship between creatinine values measured by the Jaffé method and by the enzymatic method: adjusted creatinine clearance (ml min(-1)) = creatinine clearance (ml min(-1)) x [serum creatinine (mg dl(-1))]/[serum creatinine (mg dl(-1)) + 0.2]. Subsequently, we validated this model using the data from 35 lung cancer patients. Estimated clearances of carboplatin with the original equation [creatinine clearance + 25] were systematically higher than observed clearances [mean prediction error (MPE) +/- standard error (s.e.) = 26 +/- 5%]. This positive bias was corrected by the adjustment (MPE +/- s.e. = 5 +/- 4%). When the enzymatic method is used, the adjusted creatinine clearance should be used in Calvert''s formula.