Reducing the availability of polyamines for a developing tumour.

To sustain growth and support metabolic requirements, mammals assimilate energy-producing molecules and nutrients from food. These molecules are distributed throughout the body in order to meet the requirements of the internal organs. The various demands of the different organs are to a large extent met by regulatory processes consisting of a complex interaction between hormones, growth factors and cytokines. Normal metabolic activity and partitioning of nutrients between individual organs is affected by a number of events such as stress, a limited supply of nutrients, infection or tumour growth. Since the intestine has the highest metabolic activity of all the internal organs, a tumour will initially compete with the gut for nutrients and energy-providing molecules. The polyamines represent a class of molecules where the demand in the body increases during tumour growth. A tumour can partly obtain the polyamines required to support its growth by up-regulating its own biosynthetic capacity and partly by increasing uptake from the body pool. Rather than limiting the exogenous supply of dietary polyamines we have used another approach to manipulate polyamine pools in mice. When the lectin phytohaemagglutinin is included in the diet, a fully reversible dose-dependent growth of the small intestine occurs leading to an extensive accumulation of polyamines in the intestinal epithelia. This approach of reducing the availability of exogenous polyamines to a growing tumour will be discussed.

Suppression of growth of tumour cell lines in vitro and tumours in vivo by mistletoe lectins.

A variety of studies have shown that incubation of different tumour cell lines with mistletoe lectins (MLs) in vitro has a marked cytotoxic effect. In the concentration range of low cytotoxicity cell death induced by ML-I is quantitatively due to apoptotic processes. The first events observed being membrane perforation and protusions. Simultaneous treatment of certain tumour cells with MLs rendered them more sensitive to induction of apoptosis by TNFalpha. The immunomodulatory activity of ML-I was investigated by measuring cytokine release and the results confirmed that cytokine induction by the lectin is regulated at the transcriptional level. ML-I has been shown to potentiate the effect of chemotherapeutic drugs. In addition to an in vitro effect a number of workers have demonstrated that MLs suppress tumour growth in vivo. Mistletoe lectins have been administered to animals locally to the tumour, systemic, subcutaneously or by the oral route via the diet. In many cases apoptosis was observed in the tumour and instances where complete tumour ablation has occurred have been reported. It has been hypothesized that the anticancer efficacy of tumour necrosis factor-alpha (TNFalpha) is potentiated by MLs isolated from both European and Korean mistletoe. There is accumulating evidence that both types of MLs are able to induce an anti-angiogenic response in the host suggesting that the anti-metastatic effect observed on a series of tumour cell lines in mice is in part due to an inhibition of tumour-induced angiogenesis and in part due to an induction of apoptosis.

Dietary mistletoe lectin supplementation and reduced growth of a murine non-Hodgkin lymphoma.

The growth of a murine non-Hodgkin lymphoma (NHL) tumour has been shown to be reduced by incorporating mistletoe lectin (ML-1) into the diet. The morphological characteristics of NHL tumours in mice fed ML-1-supplemented diets were different from those in LA (control)-fed mice. The degree of mitotic activity was lower and nuclear area reduced. The degree of lymphocyte infiltration was increased in tumours from ML-1 fed mice and this was accompanied by a high incidence of apoptotic bodies. Visual observation of NHL tumours from individuals fed ML-1 diet showed a poorly developed blood supply in contrast to control-fed mice. A major reduction in number of blood capillaries in NHL tumours was confirmed by microscopic evaluation of tumour sections. The results suggested an anti-angiogenic response in ML-1-fed mice. The feeding of ML-1 compared to control diet thus provided several identifiable changes in the morphology of NHL tumours which were consistent with the observed reduction in tumour weight. There was no longer histological evidence of viable tumour in 25% mice fed the ML-1 diet for 11 days. Morphological studies of the small bowel indicated (a) that the lectin induces hyperplasia, and (b) that the lectin binds avidly to lymphoid tissue of Peyer's patches. There was evidence of limited endocytosis of the lectin. An experiment where ML-3 was added to the diet of mice three days after inoculation of tumour cells showed that the lectin was able to slow down further growth of an established tumour. The results show that ML lectins induce powerful anti-cancer effects when provided by the oral route.

Anti-cancer therapy: diversion of polyamines in the gut.

The growth of a murine non-Hodgkin lymphoma (NHL) tumour, either as an intraperitoneal ascites tumour or as a solid subcutaneous tumour, has been shown to be greatly reduced by including phytohaemagglutinin (PHA), a lectin present in raw kidney bean (Phaseolus vulgaris) in the diet. The reduced rate of growth occurred in a dose-dependent manner. Based on the experimental observations it has been suggested that a competition occurs between the gut tissue undergoing hyperplasia and the developing tumour for nutrients (including polyamines) from a common body pool. This may be an important factor with regard to the observed initial low level of tumour growth following the feeding of a PHA-containing diet. Results showing that the level of hyperplasia of the small intestine in response to feeding the PHA diets was higher in non-injected mice compared to those which had been injected with tumour cells substantiated the concept of competition between gut and tumour for nutrients etc. required for growth. The observations suggest that lectins, which exhibit growth-promoting effects on the gut, may have interesting applications in the formulation of new approaches with respect to cancer treatment.

A combination of dietary protein depletion and PHA-induced gut growth reduce the mass of a murine non-Hodgkin lymphoma.

The results presented in this study show that a switch from a non-protein diet (NPD) to one of a normal protein content (LA) on the day of subcutaneous injection of non-Hodgkin lymphoma tumour cells greatly favoured the development and growth of the tumour. Interestingly, however, inclusion of the plant lectin phytohaemagglutinin (PHA) in the LA diet appeared to compete with the effect of switch to the protein-rich diet, resulting in decreased tumour size and an increased incidence of necrosis. PHA was shown to induce hyperplasia of the gut even in the presence of the growing tumour. This observation together with the fact that gut hyperplasia also occurred in animals which were fed NPD supplemented with PHA, indicated the strength of PHA as a growth signal. It would seem likely that this 'normal' growth is able to compete with the tumour for important growth factors and nutrients, including polyamines, effectively starving the tumour for these molecules and resulting in its decreased rate of proliferation.

Competition between the signal sequence and a 3'UTR localisation signal during redirection of beta-globin mRNA to the endoplasmic reticulum: implications for biotechnology.

Secretion of an intracellular protein from a cell factory requires as a first step the redirection of the mRNA for synthesis of the protein on the endoplasmic reticulum. The feasibility of retargeting a mRNA coding for an intracellular protein to the endoplasmic reticulum was investigated using Ltk- fibroblasts stably transfected with gene constructs in which rabbit beta-globin coding region and 5'UTR was linked to albumin signal sequence and different 3'untranslated regions. Globin transcripts with the native globin 3'untranslated region or with the 3'untranslated region of c-myc are present in free/cytoskeletal-bound polysomes. The addition of the signal sequence from rat albumin redirects both these globin transcripts to membrane-bound polysomes but the presence of the c-myc 3'UTR reduces the extent of redirection. Globin transcripts with both the signal sequence and 3'untranslated region from the albumin gene are efficiently redirected to membrane-bound polysomes. The results suggest competition between 5' and 3' localising signals. The addition of the signal sequence does not destabilise the mRNA nor affect translational efficiency. It is concluded that it is possible to retarget an mRNA to the endoplasmic reticulum while maintaining stability and translational capacity. This has important implications for the development of vectors to promote secretion of intracellular proteins from cell factories.

The growth of an established murine non-Hodgkin lymphoma tumour is limited by switching to a phytohaemagglutinin-containing diet.

The growth of a non-Hodgkin lymphoma, developing subcutaneously as a solid tumour in NMRI mice, is markedly diminished by including phytohaemagglutinin (PHA), a lectin present in raw kidney bean (Phaseolus vulgaris), in the diet. In the experiment described in this communication the effect of first allowing tumours to develop for 5 days before switching the mice to a diet containing PHA at different concentrations was tested to establish whether or not feeding the lectin at late times also resulted in reducing tumour growth. This switch of diet indeed proved to be effective in slowing down growth of the lymphoma tumour. The reduced rate of growth occurs in a dose-dependent manner. We have suggested that a competition between the gut epithelium undergoing PHA-stimulated hyperplasia and the developing tumour may occur for polyamines and other nutrients from a common body pool and this could be an important contributory factor with regard to the observed low level of tumour growth following the feeding of PHA-containing diet. Recent data which showed that the level of hyperplasia of the small bowel in response to feeding the PHA diets was higher in non-injected mice compared to those which had been injected with tumour cells substantiated the concept of competition between gut and tumour for nutrients and other requirements for growth.

Ribosomal proteins sustain morphology, function and phenotype in acute myeloid leukemia blasts.

Translation of mRNA is a prerequisite for cell proliferation, differentiation and viability. We have studied the effect of ribosome protein factors (GPRE) on acute myeloid leukemia (AML) blast cells. Ribosomes were isolated from MPC-11 cells using ultra-centrifugation. GPRE were extracted using a high KCl procedure. Blast cells from six AML patients were grown in suspension cultures for 24 and 96 h. GPRE or granulocyte macrophage-colony stimulating factor (GM-CSF) were added at the start of the incubation. GPRE, but not GM-CSF, prevented chromatin condensation and fragmentation of blast cell nuclei in AML-M2, -M4 and -M5 and the loss of nucleoli in AML-M2 and -M5. The fraction of phagocytosing blast cells in AML-M1, -M2, -M4 and -M5 was increased by GPRE. GPRE stimulated opsonin-dependent and -independent attachment and internalisation of N. meningitidis. GPRE increased the fraction of blasts expressing CD11b and CD32 in AML-M2 and -M5. GPRE diminished the fraction of AML-M5 cells bearing CD35 and CD32. GPRE also decreased the fraction of CD11c-bearing AML-M2 and -M5 cells. GM-CSF potentiated effects of GPRE in AML-M1, -M2, -M4 and -M5. GPRE and GM-CSF in combination affected phagocytosis and surface antigen expression in blast cells that were not influenced by either factor alone. Neither GPRE nor GM-CSF induced terminal differentiation or DNA-synthesis. We conclude that GPRE affects AML blast cell morphology, function and surface molecule expression, possibly by inhibiting apoptosis. The effects of GPRE may be mediated by ribosomal proteins that regulate translation and modulate the subcellular distribution of mRNA species.

The induction of gut hyperplasia by phytohaemagglutinin in the diet and limitation of tumour growth.

The growth of a transplantable murine non-Hodgkin lymphoma tumour, developing either intraperitoneally as an ascites tumour or subcutaneously as a solid tumour, has been shown to be markedly diminished by including phytohaemagglutinin (PHA), a lectin present in raw kidney bean (Phaseolus vulgaris) in the diet. In NMRI mice fed PHA within the range 0.45-7.0 mg/g diet, tumours which developed during a 10 day period after subcutaneous injection of cells were about 35% of the dry weight of those in lactalbumin-fed (control) animals. The reduced rate of growth occurred in a dose-dependent manner within the range 0.45-3.5 mg/g diet. Based on these observations it has been suggested that a competition between the gut epithelium undergoing hyperplasia and the developing tumour may occur for nutrients from a common body pool, and this may be an important factor with regard to the observed initial low level of tumour growth following the feeding of a PHA-containing diet. Observations which showed that the level of hyperplasia of the small bowel in response to feeding the PHA diets was higher in non-injected mice compared to those which had been injected with tumour cells substantiated the concept of competition between gut and tumour for nutrients etc. required for growth. Experiments with a second murine tumour cell line (a plasmacytoma) in Balb/c mice gave similar results indicating that the effect of PHA was not restricted to a single tumour system.

Intracellular levels of polyamines in Krebs II lymphosarcoma cells in mice fed phytohaemagglutinin-containing diets are coupled with altered tumour growth.

The number of Krebs II tumour cells recovered from the ascitic fluid of mice fed for 8 days on a lactalbumin (La) control diet was about three times higher than that in animals fed a phytohaemagglutinin-containing (PHA) diet. Feeding a PHA diet for less than 8 days after tumour cell injection also led to a reduction in tumour cell growth. There was an apparent inverse relationship between the total tumour cell count and the intracellular content of putrescine, spermidine and spermine. Hyperplasia of the small intestine occurred in the mice during the development of the ascites. A series of other organs were not affected in the same manner. The results indicate that the polyamine content of Krebs II ascites cells must increase by more than three-fold in order to achieve the intracellular concentration necessary to be able to enter the S-phase. A partial synchronization of the tumour cell population is suggested. Hyperplastic growth of the small intestine would appear to compete with tumour cells for polyamines from a common body pool.

The compartmentalization of protein synthesis: importance of cytoskeleton and role in mRNA targeting.

Following the synthesis of mRNA molecules in eukaryotic cells, the transcripts are processed in the nucleus and subsequently transported through the nuclear membrane into the cytoplasm before being sequestered into polysomes where the information contained in the RNA molecule is translated into an amino acid sequence. Recent evidence suggests that an association of mRNAs with the cytoskeleton might be important in targeting mechanisms and, furthermore, in the transport of mRNA from the nucleus to its correct location in the cytoplasm. Until recently, polysomes have been considered to exist in two classes, namely free or membrane-bound. There is now compelling evidence, however, that ribosomes, in addition to being associated with endoplasmic reticulum membranes, also are associated with components of the cytoskeleton. Thus, a large number of morphological and biochemical studies have shown that mRNA, polysomes and translational factors are associated with cytoskeletal structures. Although the actual nature and significance of the interaction between components of the translational apparatus and the cytoskeleton is not yet understood in detail, it would seem evident that such interactions are important in both the spatial organization and control of protein synthesis. Recent work has shown that a subcellular fraction, enriched in cytoskeletal components, contains polysomes and these (cytoskeletal-bound) polysomes have been shown to contain specific mRNA species. Thus, a population of cytoskeletal-bound polysomes may provide a specialized mechanism for the sorting, targeting and topographical segregation of mRNAs. In this review, current knowledge of the subcellular compartmentalization of mRNAs is discussed.

The effect of switching between a phytohemagglutinin-containing and a control diet on the growth and lipid content of a Krebs II lymphosarcoma tumor.

The present study concerns the importance of the timing of feeding mice a PHA-containing diet (7 mg g-1 diet) on tumor formation. The major decrease in tumor weight occurred in mice fed on the PHA diet for 11 days. A marked reduction was also observed in animals pre-fed for 3 days with PHA before tumor cells were injected and the diet then changed to lactalbumin, La. A large decrease in tumor weight was also evident when a change of diet from La to PHA was made on the day of tumor cell inoculation. Despite the presence of the developing tumor PHA was able to induce hyperplasia of the small intestine in all groups of animals fed PHA during a part or the whole of the experiment. The dry weights of tumors attained in each of the experimental groups plotted as a function of duration of PHA feeding, and the percentage lipid content of the tumors, mirrored almost exactly one another, suggesting that the availability of essential lipid material is severely reduced by the lectin. This would appear to have a major effect on the observed reduction in tumor growth.

Dietary phytohaemagglutinin slows down the proliferation of a mouse plasmacytoma (MPC-11) tumour in Balb/c mice.

Ten days after subcutaneous injection of MPC-11 cells, plasmacytoma tumours which developed in female Balb/c mice fed on a diet containing the kidney bean lectin phytohaemagglutinin (PHA) at a concentration of 7.0 mg g-1 diet, weighed only about 38% of those fed a lactalbumin (La) control diet. The reduction in growth caused by the lectin appeared to occur in a dose-dependent manner but the values did not reach significance before PHA was at a concentration of 7.0 mg g-1 diet. Pre-feeding with the lectin caused a further 50% reduction in tumour weight. In contrast to the reduction in tumour size the inclusion of PHA in the diet elevated the mean dry weight of the small intestine in a dose-dependent manner, values reaching significance at 3.5 mg g-1 diet. The results showed that gut hyperplasia was able to occur even in the presence of the developing tumour. A lypolytic effect of PHA occurred at high concentration. The observations suggest that PHA itself does not have a direct effect on the tumour cells, but an inter-relationship between gut hyperplasia and decreased tumour growth is indicated.

Phytohemagglutinin-induced gut hyperplasia and the growth of a mouse lymphosarcoma tumor.

NMRI mice injected subcutaneously with Krebs II lymphosarcoma cells and fed on a diet containing the kidney bean lectin phytohemagglutinin (PHA) within the range 0.45-7.0 mg/g diet, developed tumors during a 10 day period which on average were only 35% of the dry weight of tumors in lactalbumin (La) fed mice (control). The reduction in growth occurred in a dose-dependent manner in the range 0.45-3.5 mg/g diet. The degree of hyperplasia of the small intestine in response to feeding the PHA diets was higher in non-injected compared to injected mice. A lipolytic effect of PHA was observed above 1.75 mg/g diet in control mice and the highest concentration had a major effect on body weight. Since the index of hyperplasia at the lowest PHA concentration tested did not correlate with the reduction in tumor size, it is suggested that other factors in addition to the initial lectin-induced gut hyperplasia are involved in slowing down the progression of tumor growth.

Switching between control and phytohaemagglutinin-containing diets affects growth of Krebs II ascites cells and produces differences in the levels of putrescine, spermidine and spermine.

Almost twice as many ascites tumour cells were recovered from mice pre-fed for 3 days on a lactalbumin (La)-based control diet, injected with Krebs II ascites cells and then maintained on the same diet for a further 8 days, when compared with mice fed on a phytohaemagglutinin-containing (PHA) diet for the whole period. A dietary switch on the day of injection of tumour cells produced an intermediate effect; mice switched to the La diet after pre-feeding on PHA for 3 days developed somewhat more tumour cells than when the opposite dietary switch was performed. The polyamine content in the tumour cells was lowest in the mice fed on La, and highest in mice fed PHA for the duration of the experiment, respectively. Since large amounts of extraneous polyamines are required in order to sustain tumour growth, and the hyperplastic growth of the gut which occurs in response to injesting the lectin is a polyamine-dependent process, it is evident that the two growth signals compete with one another for important nutrients/growth factors, including polyamines.

Free, cytoskeletal-bound and membrane-bound polysomes isolated from MPC-11 and Krebs II ascites cells differ in their complement of poly(A) binding proteins.

A three-step detergent/salt extraction procedure (Vedeler et al., Mol Cell Biochem 100: 183-193, 1991) was used to isolate free polysomes (FP), cytoskeletal-bound polysomes (CBP) and membrane-bound polysomes (MBP) from MPC-11 and Krebs II ascites cells. Polysomes were pelleted, washed with high salt buffer and re-pelleted. Proteins in the dialysed high-salt extracts were subjected to poly(A) Sepharose chromatography and poly(A) binding and non-binding proteins were separated by SDS-PAGE. In MPC-11 cells the FP fraction contains thirteen poly(A) binding proteins and four non-poly(A) binding proteins while the corresponding fraction in Krebs II ascites cells has four poly(A) binding proteins and six proteins which do not bind poly(A). The CBP fraction isolated from MPC-11 cells has a complement of ten poly(A) binding proteins, four which are non-poly(A) binding, and a protein of 105 kDa which has both poly(A) binding and non-poly(A) binding properties. In the CBP fraction prepared from Krebs II ascites cells a protein band at 32 kDa exhibits both poly(A) binding and non-poly(A) binding properties. In this fraction there are six poly(A) binding proteins and an additional eight which do not bind poly(A). Of the total number of proteins eight of these have a molecular weight below 40 kDa. The MBP fraction in MPC-11 cells contains three poly(A) binding proteins and eleven with non-poly(A) binding properties. In contrast this fraction in Krebs II ascites cells has a complement of thirteen poly(A) binding and ten non-poly(A) binding proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

A diet containing the lectin phytohaemagglutinin (PHA) slows down the proliferation of Krebs II cell tumours in mice.

Mice injected intraperitoneally with Krebs II cells and then fed on a diet containing the lectin phytohaemagglutinin (PHA) developed ascites tumours more slowly than mice fed on a control diet. After an 8-day period following injection the number of cells recovered from mice maintained on the PHA diet was half that from those fed the control diet. A switch of diet from control to PHA on day 4 after injection resulted in a large decrease in number of tumour cells recovered. Mice injected s.c. also developed tumours at later times when fed on the PHA diet. A quantitative of ribosomes in polysome-containing fractions showed no major differences in protein synthesis in control mice and those fed the PHA diet.

The effect of insulin on proteins associated with free, cytoskeletal-bound and membrane-bound polysome populations.

It is known that insulin treatment increases the rate of protein synthesis in many cells and tissues and that it causes changes in the distribution of ribosomes between free (FP), cytoskeletal-bound (CBP) and membrane-bound polysome (MBP) populations. This paper concerns an analysis of the pattern of proteins in high-salt extracts of FP, CBP and MBP isolated from Krebs II ascites and MPC-11 cells. A combined detergent/salt extraction procedure was used to isolate the three fractions of polysomes from control cells and from cells following short-term stimulation with insulin. There were differences in the protein patterns in the individual fractions and changes occurred after insulin stimulation.

Morphological changes in Krebs II ascites tumour cells induced by insulin are associated with differences in protein composition and altered amounts of free, cytoskeletal-bound and membrane-bound polysomes.

A three-step sequential detergent/salt extraction procedure was used in order to isolate three distinct subcellular fractions containing free (FP), cytoskeletal-bound (CBP) and membrane-bound polysomes (MBP), respectively, from Krebs II ascites cells (Vedeler et al., Mol Cell Biochem 100: 183-193, 1991). The purpose was to study changes in the distribution of polysomes in these three fractions during long-term incubation with insulin under either stationary conditions or in roller suspension culture. Insulin caused a redistribution of polysomes between FP, CBP and MBP fractions. The hormone appeared to promote an entry of ribosomes into polysomes both in CBP and MBP populations. When cells were grown in stationary culture in the presence of insulin and thus promoted to attach to the substratum and undergo morphological changes, a diversion of ribosomes from CBP into MBP was observed. The level of protein synthesis was apparently very high in this latter fraction since more than 70% of ribosomes were in polysomes. Morphological changes observed following insulin treatment were accompanied by a shift of certain proteins among subcellular fractions (for example actin and p35). The fibronectin content was about 20% higher in attached compared to non-attached cells. The results suggest that morphological changes induced by stimulation with insulin are associated with an increased activity of MBP, presumably reflecting a requirement for an increased synthesis of membrane proteins.