Energy metabolism in nongrowing mice with sarcoma.

The time course of energy metabolism has been studied in weight-stable and nongrowing mice with a transplantable methylcholanthrene-induced sarcoma. Daily oxygen consumption and carbon dioxide production were measured in relation to the tumor growth from the time of tumor implantation. The time course of energy dynamics was related to the end-state changes in body composition. Freely fed sarcoma-bearing mice decreased their whole-body energy expenditure in proportion to the tumor growth. This was due to the accompanying anorexia. The alteration in oxygen consumption and carbon dioxide production was continuously evident 24 hr/day in sarcoma-bearing mice. The tumor-bearing mice lost body fat and had decreased respiratory quotient, while pair-fed controls maintained their body composition, and their respiratory quotients agreed with the food respiratory quotient. Loss of body lipids in freely fed sarcoma-bearing mice reflected a negative energy balance, accompanied with increased fat oxidation, while maintenance of body composition in pair-fed controls reflected a decreased metabolic rate. Sarcoma-bearing mice showed a significantly higher energy expenditure in relation to their food intake compared to that of pair-fed controls. Estimates of partition of oxygen uptake in sarcoma-bearing mice support that both the host and the tumor account for the elevated energy expenditure. This study has confirmed a small but significantly increased energy expenditure in sarcoma-bearing mice, which was continuously present 24 hr/day in spite of unlimited availability of food. This illustrates the fatal outcome of experimental cancer.

Protein synthesis in liver tissue under the influence of a methylcholanthrene-induced sarcoma in mice.

Amino acids are incorporated at increased rates into hepatic proteins in tumor-bearing humans and animals. In this study, we hoped to elucidate whether this is an expression of increased hepatic protein synthesis or altered isotope dilution in the precursor pool(s). Liver tissue from sarcoma-bearing mice (MCG 101) showed increased specific activities of arginine and leucine in hepatic proteins after i.p. injection of these precursors. The specific radioactivity of leucine in the free amino acid incorporation rate into proteins was also found in incubated liver slices and in a cell-free system of incubated free and membrane-attached polysomes. The increased amino acid incorporation was the net result of increased as well as decreased relative turnover of various hepatic proteins. The hepatic content of RNA was increased, but hepatic DNA and protein content was unchanged in tumor-influenced livers. Increased amino acid incorporation into hepatic proteins in tumor-bearing animals and also probably in cancer patients is due to a net increased hepatic protein synthesis, probably not confined to acute-phase reactants only.

Glucose flux in relation to energy expenditure in malnourished patients with and without cancer during periods of fasting and feeding.

Glucose dynamics, energy metabolism, and nitrogen balance were studied in eight malnourished cancer patients and seven malnourished patients without cancer. Glucose flux was measured by single injection of [6-3H]glucose and [U-14C]glucose. Energy expenditure was measured by indirect calorimetry. Each patient was studied after an overnight fast and during constant gastric infusion of a formula diet. Cancer patients had elevated glucose flux when fasting, corresponding to 42% of their spontaneous daily intake of glucose. At least one-half of the elevated flux in cancer patients compared with controls was due to increased recycling of glucose carbon after an overnight fast. Feeding doubled the total glucose flux in both cancer and control patients. The recycling was unchanged in the cancer group and disappeared in the controls during feeding. The increased glucose flux in cancer patients was concomitant with normal resting energy expenditure during periods of both fasting and feeding. Glucose flux in relation to energy expenditure was doubled in cancer patients compared to controls, and the glucose flux in fed cancer patients was similar to the rate of glucose infusion, which shows that the endogenous production of glucose was not inhibited. Cancer and control patients reached a comparable positive energy and nitrogen balance, allowing for their overall caloric intake. Our results show that cancer patients seem to have a characteristically increased glucose demand, which contributes to their weight loss when fasting. The energy drain by this elevated glucose flux can explain, as a maximum estimate, a loss of about 0.9 kg of body fat per 30-day period.

Reutilization of amino acid carbons in relation to albumin turnover in nongrowing mice with sarcoma.

Reutilization of amino acid carbons was evaluated in relation to increased turnover of albumin in tumor-bearing mice. A methylcholanthrene-induced sarcoma (MCG 101) was used in nongrowing mice (C57BL/6J). Sarcoma-bearing mice developed hypoalbuminemia, but pair-fed controls did not. The hypoalbuminemia was caused by increased albumin degradation rate, measured by injection of Na214CO3, and by exponentially increased deposition of albumin into the tumor compartment. The fractional synthesis rate of albumin was doubled in tumor-bearing mice compared with controls. The translational capacity of albumin synthesis evaluated in vitro was maintained in tumor host livers. The recycling of [14C]leucine carbons was almost extinguished in plasma albumin of sarcoma-bearing mice, while that of control mice contributed to 30 to 40% of the total leucine carbon flux in turned over albumin. The recycling of arginine carbons was also different when measured after simultaneous injection of [guanido-14C]arginine and [2,3-3H]arginine. The hepatic pool of free leucine was increased by 22% in tumor-bearing mice. It is concluded that increased albumin degradation in cancer may be a disordered event and is earlier and of initially greater quantitative importance than is altered synthesis of albumin for the development of hypoalbuminemia in experimental cancer.

Evaluation of anorexia as the cause of altered protein synthesis in skeletal muscles from nongrowing mice with sarcoma.

The importance of decreased food intake as the mechanism behind altered protein metabolism in skeletal muscle in cancer was evaluated. A methylcholanthrene-induced sarcoma (MCG 101) transplanted in weight-stable and nongrowing mice (C57BL/6J) was used as the tumor-animal model. Three study groups with appropriate control groups were used: sarcoma-bearing mice; pair-fed mice; and starved mice. The synthesis of myofibrillar and sarcoplasmic proteins was decreased in sarcoma-bearing mice. This was correlated to decreased content of RNA in the muscles and caused a net loss of muscle tissue was measured by dry weight of skeletal muscles. The incorporation rate of amino acids into myofibrillar and sarcoplasmic proteins was decreased to the same extent in the pair-fed mice as that in the sarcoma-bearing mice. This probably reflected decreased protein synthesis, since the radioactivity (dpm/mg) did not differ significantly in the crude transfer RNA fraction between the groups. Separation of soluble proteins from muscle tissue by means of ion-exchange chromatography showed that the pattern of decreased protein synthesis was not tumor specific when compared to muscle affected by starvation. The decrease in protein synthesis was more or less selective, since the synthesis of basic proteins was considerably decreased and was influenced more than were neutral and acidic proteins in both cancer and starvation. Anorexia of a tumor-bearing host is a sufficient trigger to induce decreased protein synthesis in skeletal muscles, but other factors may also be of quantitative importance.

Single-shot mega-electronvolt ultrafast electron diffraction for structure dynamic studies of warm dense matter.

We have developed a single-shot mega-electronvolt ultrafast-electron-diffraction system to measure the structural dynamics of warm dense matter. The electron probe in this system is featured by a kinetic energy of 3.2 MeV and a total charge of 20 fC, with the FWHM pulse duration and spot size at sample of 350 fs and 120 µm respectively. We demonstrate its unique capability by visualizing the atomic structural changes of warm dense gold formed from a laser-excited 35-nm freestanding single-crystal gold foil. The temporal evolution of the Bragg peak intensity and of the liquid signal during solid-liquid phase transition are quantitatively determined. This experimental capability opens up an exciting opportunity to unravel the atomic dynamics of structural phase transitions in warm dense matter regime.

Tumor cytokinetic response to total parenteral nutrition in patients with head and neck cancers.

Refeeding of patients with malignant tumors may induce tumor-cell DNA synthesis. The present study was aimed at evaluating whether induction of altered cell-cycle kinetics could be induced by intravenous total parenteral nutrition (TPN) in tumor biopsies from head and neck cancers. Nine malnourished patients with squamous cell carcinoma in the head-and-neck area were investigated before and after 5-7 d of continuous TPN. Tumor biopsies were taken in both fasted and fed states for determination of 1) ornithine decarboxylase (ODC) activity, which is rate limiting for polyamine synthesis; 2) flow-cytometric-DNA-distribution measurements; and 3) the fraction of proliferating cells expressed as immunohistochemical reactivity with the monoclonal antibody Ki-67. The histopathological differentiation, the fraction of aneuploidic cells, ODC activity, and Ki-67 reactivity were not significantly related to each other, although the number of aneuploidic cells in replicative phases correlated with the number of cells expressing the Ki-67 antigen (r = 0.86, P less than 0.01). Tumor cytokinetics showed no evidence of being changed by TPN administration.

Refeeding after fasting in the rat: energy substrate fluxes and replenishment of energy stores.

Rats were fasted and refed and concentrations of plasma glucose, glycerol, triglyceride, insulin, and glucagon as well as glucose kinetics after injection of labeled glucose and glycerol were determined. In addition, concentrations and synthesis in vivo (from tritiated water) and in vitro of glycogen, triglycerides, and protein were followed in liver, muscle, and adipose tissues. The refeeding state after fasting was characterized by a decrease in glucose and triglyceride concentrations in plasma. Glucose turnover rate was increased. Protein losses were repleted, first in the liver then in muscle tissue. Synthesis of glycogen and lipid increased above control in liver and adipose tissue. These results are compatible with an increased outflux during refeeding of different energy substrates from plasma into the depleted protein and energy stores to an extent causing lower concentration of these substrates. Such phenomena might be of importance for energy intake regulation during the phase.

Ornithine decarboxylase activity in mouse tumour tissue in response to refeeding and diet components.

Ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase activity (SAMD) were measured in tumour tissue in mice during periods of starvation (24 h) and refeeding. Starvation led to a 60% reduction in tumour ODC activity. Refeeding normalised the activity within 4 h. Restitution in ODC activity, representing de novo enzyme synthesis, preceded DNA resynthesis. SAMD activity continued to fall along the increase in ODC activity during refeeding, while difluoro-methyl-ornithine (DFMO) caused a compensatory increase in SAMD activity as expected. A fall and regain in ODC activity was associated with inhibition and regrowth of the tumour. Starvation-refeeding was not related to any decrease in tumour polyamine concentrations, while systemic DFMO blockade was. Glucose stimulated ODC when refed orally, but not when given systemically. Tumour ODC activity was not decreased in refed mice by anti-insulin, a procedure that antagonised insulin's bioactivity. Exogenous insulin did not stimulate tumour ODC activity. Our results suggest that gastrointestinal metabolism of carbohydrates stimulates the release of a factor, which initiates both ODC activity and DNA synthesis in tumour cells. This factor was not insulin.

Tumour purine nucleotides and cell proliferation in response to exercise in rats.

Voluntary physical exercise can delay the onset of anorexia and cachexia in tumour-bearing rats. A substrate deviation in the host which has been hypothesised as tumour burden is reduced despite an increase in food intake. Therefore, we determined the levels of purine nucleotides, the energy charge and the cell division rate in tumours from exercising animals in the postexercise period. Tumour content of purine nucleotides was analysed by HPLC. Tumour cell kinetics was studied by flow cytometry after incorporation of bromodeoxyuridine (BrdU) into DNA. Exercising animals demonstrated a 34.4% reduction in tumour volume (P < 0.05) but a 1.31-fold increase in energy charge in tumour tissue (P < 0.05). Labelling index (LI), DNA synthesis time (Ts) and potential doubling time (Tpot) were not significantly altered. These results suggest that the influence on tumour growth is closely related to the exercise period.

Effects related to indomethacin prolonged survival and decreased tumor-growth in a mouse-tumor model with cytokine dependent cancer cachexia.

Tumor-bearing mice with two different locally growing malignant tumors (epithelial like, MCG 101; malignant melanoma, K1735-M2) were used to evaluate the putative role of prostaglandins for survival and local tumor growth in experimental cancer. Daily systemic injections of indomethacin (1 mu g/g bw) were used to block prostaglandin production in normal and T-cell deficient tumor-bearing nude mice. Tumor progression was determined by measurements of tumor weight, DNA-synthesis, cell cycle kinetics in vivo and in vitro (flow cytometry), tumor tissue concentrations of polyamines (putrescine, spermidine, spermine) and tumor tissue gene expression of growth regulating factors (IL-1 alpha, IL-6, TNF alpha, A,B-PDGF, EGF, VEGF, bFGF, TGF beta(3), angiogenin and transferrin receptor). Tumor tissue content of von Willebrandt factor VIII was estimated by immunohistochemistry. Indomethacin had no effect on survival, host nutritional state or local tumor growth in mice bearing the malignant melanoma with low PGE(2) production. In contrast, indomethacin prolonged survival, improved cachexia and decreased tumor growth in mice bearing the MCG 101 tumor with hundredfold higher prostaglandin tumor production, leading to elevated liver and muscle tissue as well as plasma concentrations of PGE(2). Indomethacin inhibited almost completely the high tumor PGE(2) production in MCG tumors, leading to prolonged potential doubling time for tumor growth in vivo, and a trend to decreased tumor tissue concentration of polyamines (spermidine). Indomethacin had no inhibitory effect on tumor cell proliferation in vitro, although PGE(2) production was decreased by 75%. The effect of indomethacin in vivo was independent of T-cells and was observed with similar magnitude irrespective of the number of MCG cells (10(4)-10(6)) implanted or the site of implantation (s.c., i.p., liver, lung, skeletal muscles). Tumor growth inhibition by indomethacin was not intrinsically transferable by tumor cells from indomethacin treated tumor-animals. Tumor expression of mRNA for several growth regulating factors were either increased (IL-6, TNF alpha, GM-CSF, TGF beta(3)) unchanged (EGF, VEGF, PDGF A,B, IL-1 alpha, transferrin receptor) or decreased (b-FGF and angiogenin) (p<0.05) by indomethacin treatment of MCG mice. Decreased tumor content of von Willebrandt factor VIII in combination with an attenuated tumor vasculature were associated with decreased tumor growth (p<0.05). Our results confirm that high tumor production of prostaglandins was related to reduced survival. Tumor prostaglandins probably promote local tumor growth by stimulation of tumor surrounding cells to produce growth factor(s) for tumor angiogenesis including tumor and matrix cell proliferation unrelated to immune cells.

Glycerol dynamics in weight-losing cancer patients.

This study was designed to show whether weight-losing cancer patients have an elevated glycerol turnover. Four groups of patients were examined: weight-losing cancer patients, weight-losing patients without cancer, cancer patients without weight loss, and weight-stable and well-nourished hospitalized control patients. Glycerol was infused intravenously at three different rates (200, 400, and 800 mumol/hr/kg body weight) after an overnight fast. This allowed measurement of clearance and plasma glycerol turnover. Weight-losing cancer patients (group 1) had an almost threefold higher glycerol turnover per kilogram of body weight compared with malnourished and well-nourished noncancer patients. However, both malnourished cancer and noncancer patients had an elevated glycerol turnover compared with well-nourished patients when glycerol turnover was related to whole body lipids. The results how that progressive clinical cancer is associated with an elevated plasma glycerol turnover, probably indicating an increased whole body lipolysis. This may explain the loss of body fat during the development of cancer cachexia.

Effects of nandrolone propionate on experimental tumor growth and cancer cachexia.

We studied the tumor host response to excessive doses of an anabolic steroid (nandrolone propionate, 2.5 mg 20 g intraperitoneally every second day for 11 days) with respect to body composition and tumor cell kinetics in MCG 101 sarcoma-bearing mice (C57BL/6J) with progressive cachexia. Although survival and food intake were not affected, a significant weight gain was observed that was essentially attributed to water retention. Net protein content was increased only to a minor extent (15%), of which only the liver accounted for a significant part of the body compartments. Hepatic protein accumulation was obviously caused by decreased protein degradation, since hepatic RNA content was unchanged. After anabolic steroid administration, reduced histochemical staining of succinate dehydrogenase was observed in skeletal muscles rich in oxidative type 1 fibers, but it was not different from that of tumor-bearing control animals, which was also confirmed by measurements of citrate synthase and cytochrome c oxidase activities in skeletal muscle and liver tissue. The anabolic steroid had no significant effect on tumor growth in terms of weight progression, energy state, polyamine synthesis rate, cell division rate, and cell cycle cytocompartments. We conclude that anabolic steroid supplementation is not therapeutically beneficial in counteracting progressive weight loss in experimental cancer.

Multiple unrelated chromosome abnormalities in a metastatic mucoepidermoid carcinoma of the parotid gland.

We describe cytogenetic findings in a poorly differentiated, metastatic mucoepidermoid carcinoma of the parotid gland. The tumor was characterized by multiple, unrelated chromosome abnormalities. Except for two small aberrant clones showing t(1;7) and t(2;15), respectively, all other abnormal cells showed unique, mostly structural rearrangements peculiar to each cell. No less than 34 different abnormal karyotypes were observed. A similar karyotypic heterogeneity was also described recently in squamous cell carcinomas of the head and neck.

Partial 6q deletion in a human salivary gland adenocarcinoma.

G-banding analysis was performed on a cultured human salivary gland adenocarcinoma. Clonal chromosome abnormalities consisted of a 6q- marker and loss of the Y chromosome. The 6q- marker had resulted from a long arm interstitial deletion, del(6)(q22q24). The present results, together with our previous findings of 6q deletions in other types of salivary gland tumors, indicate that deletions of 6q are characteristic for the karyotypic evolution of all major types of malignant salivary gland tumors. A common pathogenetic mechanism for these tumors might well be loss of a tumor suppressor gene located on 6q.

Elective lymph node dissection in stage I cutaneous malignant melanoma of the head and neck. A report from the Swedish Melanoma Study Group.

The effect of elective lymph node dissection in patients with cutaneous malignant melanoma of the head and neck was investigated in a retrospective study. Of 517 patients in clinical stage I, 84 underwent elective dissection of the ipsilateral neck lymph nodes. In six of these patients, lymph node metastases were demonstrated at histopathological examination. There was a slight reduction in the incidence of recurrent disease in the regional lymph nodes in the group of patients who had undergone elective lymph node dissection, but this difference was not statistically significant. No significant differences were seen between the two groups regarding overall survival of disease-related survival.

Tumor suppressive action of indomethacin is NK-cell-independent.

This study was undertaken to determine whether NK-cells constitute a necessary mediator for the suppression of tumor growth by indomethacin. C57Bl mice with a methylcholantrene (MCG 101) tumor were studied. Indomethacin treatment was provided by daily subcutaneous injections (1 microgram/g body weight). NK-cells were depleted by treatment with a monoclonal antibody to NK1.1. Consecutive indomethacin injections prolonged survival in tumor bearing animals. Indomethacin was equally effective in animals with intact NK-cells as in NK-cell-depleted animals. Further, the MCG cells were apparently insensitive to the lytic activity of NK-cells in vivo. Thus, the clearance of intravenously injected MCG cells from lungs was not affected by depletion of NK-cells in vivo; in contrast, the corresponding clearance of NK-cell-sensitive YAC-1 lymphoma cells was strikingly reduced by the depletion of NK-cells. Our data suggest that NK cells are not a necessary mediator for the suppression of tumor growth by indomethacin.

Laminin-5 as a predictor of invasiveness in cancer in situ lesions of the larynx.

Squamous epithelial cancer in situ (CIS) of the upper aerodigestive tract is a histopathologically well-defined condition. There is yet no reliable way to predict whether a CIS lesion will progress to invasive cancer, remain stable or regress. In the search for markers able to foretell clinical outcome, we performed immunohistochemical staining with a polyclonal antibody against recombinant gamma 2 chain of laminin-5 in 33 laryngeal CIS lesions. All six CIS lesions which progressed to invasive cancer, within a follow-up time of 5 years, were laminin-5 positive (100%), whereas only 10 out of 27 lesions which did not progress were positive (37%) (p < 0.01). Our data showed that a positive laminin-5 laryngeal CIS lesion indicates a high risk for progression to invasive cancer.

Evaluation of ornithine decarboxylase activity as a marker for tumor growth rate in malignant tumors.

Ornithine decarboxylase (ODC) is a rate-limiting enzyme in the synthesis of polyamines. Polyamines regulate DNA synthesis by a mechanism that is not fully understood. High levels of polyamines and ODC activity are associated with rapid cell growth, particularly in tumor tissues. The aim of this study was to determine whether ODC activity as a marker for rapid alterations in tumor growth could be used to investigate whether nutritional support in cancer patients stimulates tumor cell proliferation. Weight-losing head and neck cancer patients and tumor-bearing mice (MCG 101, C57/BL) were studied during different feeding regimens. The ODC activity in tumor tissue was investigated in relation to the following variables: (1) histopathologic differentiation; (2) DNA content; and (3) bromodeoxyuridine (BrdUrd) incorporation into DNA. After the animals were starved for 24 hours, a significant reduction of tumor growth was demonstrated in the experimental tumor along with a reduction of ODC activity, an accumulation of cells in the G0G1 phase, and a reduction of cells incorporating BrdUrd into DNA. Refeeding after 24 hours generated a response by all variables. Tumor biopsy specimens from patients with head and neck cancer malignancies demonstrated aneuploidy in the cells of 70% of the patients. High ODC activity in tumor tissue was demonstrated mainly among poorly differentiated tumors, and ODC activity was correlated with the compartment size of aneuploidic cells in the tumor. High ODC activity indicated a poor short-term survival (1 year). It was concluded that experimental tumor growth is highly dependent on host feeding. However, there was no evidence supporting the claim that nutritional support to cancer patients stimulates tumor cell proliferation. Determination of ODC activity may be used to monitor rapid changes in DNA synthesis and may have prognostic significance for survival.

Proliferative pattern of head and neck cancer.

Tumor growth is primarily dependent on the fraction size of growing cells, their growth and proliferative rate, and the fractional cell death. In the present study, we focused specifically on the proliferative characteristics of squamous cell carcinomas of the head and neck region by using monoclonal antibodies and immunohistochemical methods. We studied two normally occurring antigens representative of cell proliferation: (1) ribonucleotide reductase, which is an independent cytoplasmatic enzyme and is intimately integrated in DNA synthesis, and (2) Ki-67, which is a nuclear antigen being expressed only in replicative cells. We also used intravenously injected bromodeoxyuridine (BRDU) for specific detection of tumor cells in the S phase of the cell cycle. In addition, in vivo injections of BRDU were also given to tumor-bearing mice to illustrate tumor cell kinetics by means of flow cytometry. The main observation was morphologic heterogeneity, with a high frequency of proliferative cell clusters in the cancer specimens interspersed among quiescent cells, which was demonstrated by the three monoclonal antibodies independent of each other. The experimental studies clearly visualized the transfer of BRDU throughout the tumor cell cycle. We conclude that immunohistochemical analysis provides valuable qualitative information on the proliferative pattern of tumor growth and, together with dynamic flow cytometry, may improve the clinical basis for individualized management of the cancer patient.