Effect of age and sex on lung-colony-forming efficiency of injected mouse tumour cells

The i.v. injection of a specified number of cells of either an Ehrlich ascites tumour (ELD) or spontaneous mouse mammary adenocarcinomas (MA) into C3H mice yielded a number of lung colonies which varied significantly with the age or sex of recipient mice. The yield was higher in mice of 71 weeks than in those of 15 weeks, except for MA cells injected into females, when the yield was higher in the younger mice. Sex did not influence very significantly the yield of colonies from ELD cells; in the case of MA cells the direction of sex differences depended on age. A difference in the effect of pre-immunization with age was not observed.     

Experimental study of the effect of intra-abdominal pressure during laparoscopy on tumour growth and port site metastasis

BACKGROUND: The influence of raised intraperitoneal pressure during laparoscopy on tumour growth and port site metastasis is still unknown. METHODS: Tumour growth of colonic adenocarcinoma DHD/K12/TRb was measured after laparoscopy with carbon dioxide at different pressures (0, 5, 10 and 15 mmHg) in a rat model. Cell kinetics were determined after incubation with carbon dioxide (0, 5, 10 and 15 mmHg) in vitro (n=60). Additionally, tumour growth was measured subcutaneously and intraperitoneally 4 weeks after laparoscopy at different intraperitoneal pressures (5, 10 and 15 mmHg) (n=100). RESULTS: In vitro tumour growth decreased significantly after incubation with carbon dioxide at 10 and 15 mmHg compared with a pressure of 0 or 5 mmHg. In vivo, mean(s.d.) intraperitoneal tumour weight was significantly increased after laparoscopy at 5 mmHg (919(1085) mg) and at 10 mmHg (1274(1523) mg) (P< 0.05), but decreased again after laparoscopy with an intraperitoneal pressure of 15 mmHg (731(929) mg) compared with the control group (365(353) mg) (P=0.3). Mean(s.d.) subcutaneous tumour growth was promoted after laparoscopy at 5 mmHg (172(234) mg), at 10 mmHg (190(253) mg) and at 15 mmHg (178(194) mg) compared with controls (48(33) mg) (P < 0.05). CONCLUSION: In vitro, raised intraperitoneal pressure leads to suppression of tumour growth. In vivo, intraperitoneal tumour growth is suppressed only by higher pressure (15 mmHg). Subcutaneous tumour growth is stimulated by carbon dioxide independently of the intraperitoneal pressure.     

Suppressed tumor growth and metastasis by vitamin A + BCG in Lewis lung tumor bearing mice

The anti-tumor effect of vitamin A and/or BCG was investigated in Lewis lung tumor system. Tumor growth and lung metastases were significantly suppressed, when tumor cells were mixed with BCG and inoculated subcutaneously into vitamin A-treated animals. Survival time was also prolonged by the same treatment. Vitamin A alone, without BCG, showed no effect on tumour growth, lung metastases or survival time.     

Inactivation of ribonucleotide reductase in tumour cells and inhibition of tumour cell growth by p-alkoxyphenols

A significant correlation between the inactivation of the growth-regulating enzyme ribonucleotide reductase (RR) with the growth inhibition of four different tumour cell lines has been found for seven different p-alkoxyphenol derivatives with varying lengths of alkyl side chain. In Novikoff hepatoma and human leukaemia cells, inactivation of RR by p-alkoxyphenols was monitored by electron paramagnetic resonance (EPR) spectroscopy of the catalytically essential tyrosyl radical in the subunit R2 of RR. A significant inhibition of cellular growth of Novikoff hepatoma cells, human leukaemia cells and two human melanoma cell lines (MeWo and M5) by p-alkoxyphenols was also observed by growth inhibition assays. Inactivation of RR in whole tumour cells as well as inhibition of cellular growth of tumour cell lines by p-alkoxyphenols both show an increase in inhibition with increasing length of the alkyl side chain; the most effective inhibitors are p-isobutoxyphenol, p-butoxyphenol and p-propoxyphenol. The enzyme RR, and in particular the catalytically essential tyrosyl radical in the active site, is recognized as an important cellular target for growth inhibition of Novikoff hepatoma cells, human leukaemia cells and melanoma cells by p-alkoxyphenols. Thus, the most potent RR inhibitors-p-isobutoxyphenol, p-butoxyphenol and p-propoxyphenol-may be considered as future antiproliferative drugs for the systemic treatment of melanoma as well as leukaemia and possibly other malignancies.     

Destruction of allogeneic tumour cells by peritoneal macrophages

The population of peritoneal macrophages from mice immunised with allogeneic tumor cells contains two types of cytotoxic cell. One lyses the specific target cell, the other initiates activation of macrophages and hence leads to inhibition of growth of the specific target cell. The yield of lytic effector macrophages was found to depend on the route and the nature of the cell used for immunisation and the condition of the mice. The yield correlated with the yield of complement-dependent cytotoxic antibodies. In contrast, production of specific "activator" macrophages did not depend critically on these factors. The results underline the difference between the two types of cell and suggest that they are produced independently of one another.     

1H MRS markers of tumour growth in intrasplenic tumours and liver metastasis induced by injection of HT-29 cells in nude mice spleen

We have characterized, by in vitro magnetic resonance spectroscopy (MRS), the metabolite pattern of perchloric acid (PCA) extracts of intrasplenic tumours and hepatic metastasis, produced by intra-spleen injection of the human colorectal carcinoma cell line HT-29 and its metastatic variant HT-29 MMM into nude mice. Our aim was to gain further understanding of colorectal tumour metabolism as a basis for future in vivo studies of human colon cancer by 1H MRS. Metabolite PCA extract analysis showed a good reproduction of the spectral pattern observed in human primary colon tumours, while they were very different from the spectral pattern of the host tissues (spleen and liver). The main differences between host and tumour tissues involved taurine, phosphocholine (PC), phosphoethanolamine (PE), creatine, glycogen and glucose. Creatine is the most promising marker to follow tumour growth because of its practical absence in the nude mice host tissues. Detection of variable levels of this compound and of taurine in hepatic foci in man, are suggested as possible diagnostic markers. No correlation could be found between spectral pattern differences and the different ability to metastasize of the two HT-29 cell lines used. Furthermore, indirect evidence for a functional link between taurine and myo-inositol in colon tumour cells is presented. In summary, our data suggest that the nude mice model may be a suitable system for the MRS study of the changes taking place in host tissues upon tumour progression.     

Conformational changes of the smooth endoplasmic reticulum are facilitated by L-glutamate and its receptors in rat Purkinje cells

Following a unilateral lesion of the visual cortex (cortical areas 17, 18, and 18a) in adult rats, neurons in the ipsilateral dorsal lateral geniculate nucleus (LGN) are axotomized, which leads to their atrophy and death. The time course of this neuronal degeneration was studied quantitatively, and the astroglial response was examined with glial fibrillary acidic protein immunohistochemistry. More than 95% of the neurons in the ipsilateral LGN survive during the first 3 days following a lesion of the visual cortex. However, in the next 4 days, massive neuronal death ensues, reducing the number of surviving neurons to approximately 33% of normal by the end of the first postoperative week. Between 2 weeks and 24 weeks postoperatively, the number of neurons present in the LGN declines very gradually from 34% to 17% of normal. Three days after a lesion of the visual cortex, the mean cross-sectional areas of ipsilateral LGN neurons are 13% smaller than normal (87%). By 1 week after the operation, surviving LGN neurons have atrophied to 66% of their normal area. Subsequently, the size of surviving neurons declines slowly to approximately 50% of normal at 24 weeks after the cortical lesion. Astrocytes in the ipsilateral LGN also react to cortical damage. At 1 day after a lesion of the visual cortex, glial fibrillary acidic protein immunoreactivity in the LGN is almost undetectable, but a distinct increase in immunoreactivity is seen at 3 days. Immunoreactivity peaks between 1 week and 2 weeks postoperatively and, thereafter, remains intense for at least 24 weeks. Thus, following a lesion of the visual cortex, the somata of neurons in the LGN remain essentially normal morphologically for about 3 days before the onset of rapid atrophy and death. Moreover, most of the neural cell death that occurs in the LGN after axotomy takes place in the last half of the first postoperative week.     

Dissection of tumour and host cells from target organs of metastasis for testing gene expression directly ex vivo

We report on a new methodology which allows the direct analysis ex vivo of tumour cells and host cells (lymphocytes, macrophages, endothelial cells) from a metastasised organ (liver or spleen) at any time point during the metastatic process and without any further in vitro culture. First, we used a tumour cell line transduced with the bacterial gene lacZ, which permits the detection of the procaryotic enzyme beta-galactosidase in eukaryotic cells at the single cell level thus allowing flow adhesion cell sorting (FACS) analysis of tumour cells from metastasised target organs. Second, we established a method for the separation and enrichment of tumour and host cells from target organs of metastasis with a high viability and reproducibility. As exemplified with the murine lymphoma ESb, this new methodology permits the study of molecules of importance for metastasis or anti-tumour immunity (adhesion, costimulatory and cytotoxic molecules, cytokines, etc.) at the RNA or protein level in tumour and host cells during the whole process of metastasis. This novel approach may open new possibilities of developing strategies for intervention in tumour progression, since it allows the determination of the optimal window in time for successful treatments. The possibility of direct analysis of tumour and host cell properties also provides a new method for the evaluation of the effects of immunisation with tumour vaccines or of gene therapy.     

Glucocorticoid receptor binding in proliferating and differentiating cells: modulation by a tumour promoting agent

The effects of the tumour-promoting agent, phorbol 12-myristate 13-acetate, on dexamethasone binding and protein kinase C in proliferating and differentiating murine erythroleukemic cells were investigated. Concentration dependent changes in steroid binding were observed in proliferating and differentiating cells treated with phorbol 12-myristate 13-acetate for 2h or 24h. Ligand binding in differentiating cells was consistently lower than in proliferating cells. Reduced expression of protein kinase C was observed in differentiating cells as compared with proliferating cells; the amount of enzyme protein was reduced on treatment of cells with phorbol ester.     

Clonal extinction of myelomonocytic leukemic cells by serum from mice injected with endotoxin

Culture of WEHI-3B myelomonocytic leukemic cells in semi-solid agar medium containing post-endotoxin serum led to the development of maturing granulocytes and macrophages in most leukemic colonies. Colony size was consistently increased but the colony content of colony-forming cells (stem-cell self-replication) was markedly reduced. Serial recloning of WEHI-3B colony cells in the continuous presence of post-endotoxin serum led to clonal extinction of the leukemic cells in five of seven experiments. These effects of post-endotoxin serum on WEHI-3B cells were not seen in clonal cultures of 10 other tumor lines. Serum with the capacity to induce differentiation in WEHI-3B cells could be induced by the injection of as little as 0.1 microgram endotoxin and by purified bacterial cell-wall preparations. Serum activity reached peak levels 3 - 6 h after endotoxin injection and returned to preinjection levels within 48 h.     

Intravenously injected radiolabelled fatty acids image brain tumour phospholipids in vivo: differential uptakes of palmitate, arachidonate and docosahexaenoate

This paper investigates the incorporation of intravenously (i.v.) administered radiolabelled fatty acids--[9,10(3)-H]palmitate (3H-PA), [1-14C]arachidonate (14C-AA) and [1-14C]docosahexaenoate (14C-DHA)--into intracerebrally implanted tumours in awake Fischer-344 rats. A suspension of Walker 256 carcinosarcoma tumour cells (1 x 10(6) cells) was implanted into the right cerebral hemisphere of 8- to 9-week-old rats. Seven days after implantation, the awake rat was infused i.v. for 5 min with 3H-PA (6.4 mCi/kg), 14C-AA (170 microCi/kg) or 14C-DHA (100 microCi/kg). Twenty minutes after the start of infusion, the rat was killed and coronal brain sections were obtained for quantitative autoradiography and histology. Each fatty acid showed well-demarcated incorporation into tumour tissue. Areas of necrosis or haemorrhage showed no or small levels of incorporation. The ratios of incorporation into the tumour to incorporation into contralateral brain regions were 2.8-5.5 for 3H-PA, 2.1-3.3 for 14C-AA and 1.5-2.2 for 14C-DHA. The mean ratios differed significantly between the fatty acids (P < 0.01). 3H-PA was not incorporated into necrotic tumours despite the presence of an open blood-tumour barrier, indicated by extravasated horseradish peroxidase. The incorporation rate constant of 3H-PA was similar for small intracerebral and large extracerebral tumours. The results show that 3H-PA, 14C-AA and 14C-DHA are incorporated more readily into tumour tissue than into brain, and that the increase is primarily due to increased utilization of fatty acids by tumour cells and not due to a high blood-tumour permeability. The relative increases in rates of incorporation for the different fatty acids may be related to lipid composition of the tumour and to the requirement of and specific role of these fatty acids in tumour cell growth and division.     

Sequential steps in hematogenous metastasis of cancer cells studied by in vivo videomicroscopy

Understanding metastatic spread of cancer is of upmost importance to developing successful strategies to treat this disease. In this review, we present a picture of the process of hematogenous metastasis from the initial arrest of cancer cells, their extravasation, postextravasation migration, and their replication to form tumors, based on experimental results using in vivo videomicroscopy. The cancer cells are initially arrested by size constraints within minutes of entering the circulation and with little hemodynamic destruction. Within 24-48 h >80% of these cancer cells extravasate as single cells by adhesion to and spreading along the vessel wall, often using pseudopodial projections to move into the surrounding tissue without disrupting the microcirculation. Some of the extravasated cells also use pseudopodial projections to migrate to specific structures in the tissue where they can replicate. Many cancer cells can persist as dormant cells, neither dividing nor undergoing apoptosis. Only a small fraction of extravasated cells begin to divide to form micrometastases, and only a very small fraction of these micrometastases continue to grow to form tumors. Possible clinical implications are that (1) initial arrest and extravasation may be difficult to prevent and thus may be poor therapeutic targets; (2) dormant single cells will not be affected by conventional cancer therapies which are designed to treat actively growing cells; and (3) regulation of growth of cells after extravasation is key to determining whether clinically evident metastases form - this stage of metastasis thus offers promising targets for new antimetastasis drugs.     

Recombination of protein domains facilitated by co-translational folding in eukaryotes

The evolution of complex genomes requires that new combinations of pre-existing protein domains successfully fold into modular polypeptides. During eukaryotic translation model two-domain polypeptides fold efficiently by sequential and co-translational folding of their domains. In contrast, folding of the same proteins in Escherichia coli is posttranslational, and leads to intramolecular misfolding of concurrently folding domains. Sequential domain folding in eukaryotes may have been critical in the evolution of modular polypeptides, by increasing the probability that random gene-fusion events resulted in immediately foldable protein structures.     

Vanilloid receptor agonists potentiate the in vivo local anesthetic activity of percutaneously injected site 1 sodium channel blockers

BACKGROUND: Capsaicin, the pungent ingredient in chili peppers, is a vanilloid with noxious and analgesic effects that inhibits tetrodotoxin-resistant sodium currents. Because tetrodotoxin-resistant currents are found primarily in small-diameter nociceptor afferents of the peripheral nerves, their inhibition may lead to selective analgesia. Therefore, the authors evaluated the interactions between tetrodotoxin, a site 1 sodium channel blocker, and capsaicin on nerve blockade in vivo. METHODS: Percutaneous sciatic nerve injections with 0 to 9.9 mM capsaicin, 0 to 120 microM tetrodotoxin, or both were administered to male Sprague-Dawley rats. Thermal nociceptive and motor blockade were measured. Data were expressed as medians with 25th and 75th percentiles. RESULTS: Capsaicin produced a transient increase in thermal latency with no effect on motor strength. Tetrodotoxin reduced motor strength for a longer duration than nociception. The interaction between tetrodotoxin and capsaicin was synergistic, as evidenced by (1) supraadditive prolongation of both nociceptive and motor block, with the effect of capsaicin reversed by the vanilloid antagonist capsazepine, and (2) synergism in the frequency that rats achieved maximal block shown by isobolographic analysis. The combination of tetrodotoxin and capsaicin showed less motor predominance than tetrodotoxin did alone. Similar interactions were found between tetrodotoxin and resiniferatoxin (another vanilloid), and between capsaicin and saxitoxin (another site 1 sodium channel blocker), but much less so between bupivacaine and capsaicin. CONCLUSIONS: Site 1 sodium channel blockers and vanilloids have synergistic effects on nerve blockade in vivo. These interactions may be useful in developing prolonged local anesthetics and elucidating mechanisms of functionally selective nerve blockade.     

Induction of antigen presentation by macrophages after phagocytosis of tumour apoptotic cells

Due to their resistance to classical chemotherapies, most human colorectal cancers have a high incidence and a poor prognosis. Immunotherapy using interleukin 2 (IL2) has provided disappointing results in the treatment of these cancers. Recently, however, we have demonstrated that a treatment combining a cell-differentiating agent, sodium butyrate (NaBut) with IL2 resulted in a remission of established peritoneal colorectal carcinomatosis in rats. Separately, neither NaBut nor IL2 treatment cured these tumour-bearing rats. NaBut is known to induce cell differentiation and subsequent apoptosis in epithelial cells, while IL2 stimulates the immune cells capable of participating in tumour rejection. We postulated that the significant therapeutic effect of NaBut/IL2 treatment could be attributed to a NaBut-induced increase in the immunogenicity of the cancer cells. We report here that NaBut induced an apoptotic process in rat colon tumour cells in vivo and in vitro. We observed, in an efficient cure, colocalization of apoptotic bodies and monocytes/macrophages at the periphery of the tumour. We propose that these apoptotic bodies are phagocytosed in vivo by the macrophages. We also showed in vitro that a subpopulation of macrophages involved in the phagocytic clearance of apoptotic cells expresses cell surface molecules associated with antigen presentation and stimulates the proliferation of naive splenocytes. Our data suggest that therapies that recruit massive induction of the apoptotic process in tumour cells could favour tumour antigen presentation via their specific phagocytosis by antigen-presenting cells (APCs). We propose that the development of specific therapies that stimulate both tumour cell apoptosis and the immune system could offer new opportunities in anti-cancer treatments of poorly immunogenic cancer cells.     

Ultrastructural changes in the alveolar cells of rats injected with Cerastes cerastes cerastes venom

Ultrastructural changes in the alveolar tissue of rats intraperitoneally injected with the Cerastes cerastes cerastes venom were studied in 2 different experimental groups. In the first group, each rat was given 0.73 mg/Kg as a single dose and sacrificed after 24 hours. In the second group, each rat was given a daily dose of 0.42 mg/Kg for 7 days and sacrificed 24 hours after the last injection. Proliferative changes were seen in type II alveolar cells, fibroblasts, lymphocytes, and macrophages. Type II alveolar cells of the lungs developed a large number of surfactant granules. In the 24-hour-envenomated rats, type I alveolar cells displayed swollen nuclei and masses of dilated endoplasmic reticulum. In the 7-day-treated rats, several plasma cells, adjacent interstitial cells as well as alveolar brush cells, with their characteristic short microvilli, were detected. Large masses of collagen and elastic fibers were also located in the vicinity of the alveolar brush cells and type II alveolar cells. These histopathological changes may be attributed to the body-immune response and possibly to the development of hyperplasia due to venom-induced trauma.     

Histopathology of the seminiferous tubules in mice injected with syngeneic testicular germ cells alone

Previously, a model of murine experimental autoimmune orchitis was produced by active immunization with viable syngeneic testicular germ cells without resorting to any adjuvants. The histological mode of the spermatogenic disturbance of this autoimmunity was investigated in A/J mice. A significant spermatogenic disturbance was consistently induced after the appearance of inflammatory cell responses around the tubuli recti. It first appeared seminiferous epithelium adjacent to the tubuli recti, then spread to the peripheral epithelium. The histopathology of the seminiferous tubules in the early phase ranged from partial degeneration and depletion of all kinds of germ cells to complete loss of germ cells other than some remaining spermatogonia, while both Sertoli cells and the basal lamina of the tubules appeared intact. In the late phase, depletion of Sertoli cells, disorganization of the seminiferous tubular wall or filling with many round-shaped degenerating germ cells, appearance of malformed spermatids with signet ring nuclei, depletion of immature germ cells with remaining elongated spermatids, or complete loss of the seminiferous epithelium were observed in addition to the early histopathological features.