Cytomegalovirus: an unlikely ally in the fight against blood cancers?

Cytomegalovirus (CMV) infection is a potentially fatal complication in patients receiving haematopoietic stem cell transplantation (HSCT), but recent evidence indicates that CMV has strong anti-leukaemia effects due in part to shifts in the composition of natural killer (NK) cell subsets. NK cells are the primary mediators of the anti-leukaemia effect of allogeneic HSCT, and infusion of allogeneic NK cells has shown promise as a means of inducing remission and preventing relapse of several different haematological malignancies. The effectiveness of these treatments is limited, however, when tumours express human leucocyte antigen (HLA)-E, a ligand for the inhibitory receptor NKG2A, which is expressed by the vast majority of post-transplant reconstituted and ex-vivo expanded NK cells. It is possible to enhance NK cell cytotoxicity against HLA-Epos malignancies by increasing the proportion of NK cells expressing NKG2C (the activating receptor for HLA-E) and lacking the corresponding inhibitory receptor NKG2A. The proportion of NKG2Cpos /NKG2Aneg NK cells is typically low in healthy adults, but it can be increased by CMV infection or ex-vivo expansion of NK cells using HLA-E-transfected feeder cells and interleukin (IL)-15. In this review, we will discuss the role of CMV-driven NKG2Cpos /NKG2Aneg NK cell expansion on anti-tumour cytotoxicity and disease progression in the context of haematological malignancies, and explore the possibility of harnessing NKG2Cpos /NKG2Aneg NK cells for cancer immunotherapy.

Arglabin-DMA, a plant derived sesquiterpene, inhibits farnesyltransferase.

Arglabin [1(R),10(S)-epoxy-5(S),5(S),7(S)-guaia-3(4),11(13)-dien-6, 12-olide], a sesquiterpene gamma-lactone is isolated from Artemisia glabella, a species of wormwood endemic to the Karaganda region of Kazakstan. The compound has been modified to render it water-soluble through addition of a dimethylaminohydrochloride group to the C(13) carbohydride moiety to yield Arglabin-DMA. Arglabin-DMA is a registered antitumor substance in the Republic of Kazakstan. Previously, we have shown that this compound prevents protein farnesylation without altering geranylgeranylation. We now report that Arglabin-DMA inhibits the incorporation of [(3)H]farnesylpyrophosphate into human H-ras protein by FTase with an IC(50) of no greater than 25 microM. Kinetic studies show that the phosphorylated form of this compound competitively inhibits the binding of farnesyl diphosphate to FTase. This mechanism of action is different from other reported peptidomimetic FTIs which lower the affinity of ras protein to FTase. Our in vitro studies confirm that Arglabin-DMA inhibits post-translational modification of ras protein in cells. Arglabin-DMA inhibits anchorage-dependent proliferation of NB cells (IC50=10 microg/ml) and inhibits anchorage-independent growth of NB and KNRK cells with about the same IC(50). Soft-agar colony formation assay of H-ras and K-ras transformed cells show IC(50)s to be 2 and 5 microg/ml, respectively. In primary cultures of human tumor cells, Arglabin-DMA inhibits cell proliferation of a variety of tumor types with IC(90)s in the range of 0.85 to 5.0 microg/ml. Because of these pharmacologic properties, we propose that Arglabin-DMA is suitable for the treatment of ras related malignancies.

Free and total ion concentrations in pig digesta.

Mineral bioavailability is related to the activity of the free ion or small-ligand metal ion complexes present in gastrointestinal (GI) tract digesta. Therefore, it is necessary to distinguish between total ion and free-ion/small-ligand complexes (referred to simply as "free") concentrations. Free and total cation concentration in pig digesta from various GI locations were determined. Free ions were operationally defined as those that passed through a 1,000 molecular weight cutoff filter. To test the effect of dietary supplementation on free ion concentrations, pigs were fed either basal diets of corn bran, corn grits, and soybean meal (10, 67, and 20 weight percent, respectively) or basal diets containing added Ca, Zn, Fe, and Cu. In addition, the Ca and K content of corn bran fragments retrieved from digesta was determined by energy dispersive x-ray analysis to examine whether this dietary fiber preferentially absorbed minerals, thus reducing mineral bioavailability. Free cation concentrations, expressed as a percentage of the total, averaged over all locations for both diets were: Na, 86%; K, 96%; Ca, 11%; Mg, 40%; Zn, 5%; Fe, 4%; and Cu, 11%. For Ca, Mg, Zn, and Cu, the free:total cation concentration ratios differed (P < .05) between upper and lower GI tract. Mineral supplementation did not alter free:total ratios of any ion in the GI tract. For supplemented diets, mineral concentrations generally were higher throughout the GI tract, as were concentrations of free Ca. Free concentrations of Zn and Cu in the jejunum and ileum were higher (P < .01) with supplemented diets.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell proliferation kinetics of normal and tumour tissue in vitro: quiescent reproductive cells and the cycling reproductive fraction.

Current methods for measuring the cell kinetics of human tumours are made and interpreted within the context of a simplistic two compartment model for cell proliferation, consisting of cells that are cycling and those that are not. It is now recognized that the non-cycling compartment of many tumours is heterogeneous, composed of non-reproductive end-stage cells and reproductive cells that are dormant/quiescent. We have developed an in vitro analysis that distinguishes for the first time quiescent reproductive cells from non-reproductive end-stage cells and have integrated this analysis with monolayer clonogenic and suicide assays to simultaneously quantitate the duration of the cell cycle and reproductive cells that are: cycling, quiescent, clonogenic, and non-reproductive end-stage cells. We have defined a new parameter, the Cycling Reproductive Fraction (CRF), which is the cycling cell population referenced specifically to the reproductive cell population. Measurements of CRF from 72 tumour biopsies and from 5 normal foreskins showed that CRF approached 100% in some tumours; however, CRF showed near normal values (< 1%) in others suggesting that cell cycle control may be maintained in some tumours. Because of CRF's improved specificity, we believe that CRF may enhance classification, prognostication, and the optimization and prediction of response to chemotherapy.

Adverse response to vital bleaching.

A case study is presented that described an acute flare-up of a tooth following a vital bleaching procedure. The case illustrates (a) the importance of assessing the pulpal and periradicular status of a tooth prior to any vital bleaching procedure; and (b) the alteration of the local adaptation syndrome by the bleaching agent.

Cellular radiosensitivity of primary head and neck squamous cell carcinomas and local tumor control.

The low dose survival parameters of human tumor cell lines have been shown to correlate with the perceived clinical radiosensitivity of different tumor histologic types. This conclusion has been generated from the analysis of a large number of cell lines and has, therefore, served as the basis for attempts to develop predictive assays of tumor radiocurability. In this study, the tumors from 72 patients with head and neck squamous cell carcinoma have been grown in an adhesive tumor cell assay system and their sensitivity to radiation has been measured. All patients in this study were treated with post-operative radiotherapy, the surgical margins were negative, and any patient that had received chemotherapy was excluded. The average S2 (survival at 2.0 Gy) value of the 72 cultures was 0.33, with the values ranging from 0.11 to 0.91. All patients were evaluated for local tumor control. They have been followed for about 1 year and continued follow-up is still in progress. The average survival at 2.0 Gy of cultures derived from the 12 patients that have had recurrences so far is slightly higher (0.40) than that from those who appear so far to have local tumor control (0.30). Although the general trend is that recurrent tumors yield primary cultures that are slightly more resistant, the difference is not statistically significant.

Radiosensitivity of human head and neck squamous cell carcinomas in primary culture and its potential as a predictive assay of tumor radiocurability.

The intrinsic radiosensitivity of human tumor cell cultures correlates with the clinical radiosensitivity of several different tumor histologies, as evidenced by analyses of low dose parameters of radiation survival curves generated from a large number of cell lines. Such radiosensitivity has therefore served as a basis of attempts to develop predictive assays of tumor radiocurability. In this study, the tumors from 72 patients with head and neck squamous carcinoma have been grown in an adhesive tumor-cell assay system and radiosensitivity (S2: survival at 2.0 Gy) values have been measured. The characteristics of these cultures, such as growth rate, clonogenicity and growth enhancement by epidermal growth factor, do not correlate with S2. The average S2 value of the 72 cultures is 0.33, which is lower than for cultures derived from melanomas and lung adenocarcinomas. Twenty-six patients followed up for at least 15 months have been evaluated for local tumor control. The average S2 value of the seven patients with recurrences in this group is slightly higher (0.43) than that from the other patients (0.30). There is considerable overlap of S2 values in the two groups, and more patients must be evaluated before the groups can be compared statistically.

In vitro activity of bleomycin, tallysomycin S10b, and liblomycin against fresh human tumor cells.

The objective of this study was to compare the relative in vitro cytotoxicity of bleomycin to that of two newer-generation analogues, tallysomycin S10b and liblomycin. The latter compound is of particular interest as it has recently been shown in preclinical studies to be free of a potential to cause pulmonary injury and yet to possess only a minor potential to produce myelotoxicity. Using the adhesive tumor cell culture system, we evaluated the activity of these three drugs against a panel of 13 human tumors of various types. The range of concentrations chosen was determined and normalized using a nonleukemic permanent mouse hematopoietic progenitor cell line. Those drug concentrations achieving 90% inhibition of growth (IC90) against the murine cell line were: 6.11 microM bleomycin; 7.53 microM tallysomycin S10b; and 0.6 microM liblomycin. When tested against fresh human tumors at equally myelotoxic IC90 concentrations, bleomycin and tallysomycin S10b (nonmyelotoxic compounds) both achieved 90% growth inhibition of all tumors, while liblomycin (a myelotoxic compound) produced an IC90 inhibition in 69% of all tumors. A comparison of drug IC90 values against individual fresh tumors indicated a correlation between bleomycin and its structurally related analogue tallysomycin S10b. No such correlation, however, was seen with liblomycin in comparison to either bleomycin or tallysomycin S10b. The relative activity of liblomycin versus that of bleomycin and tallysomycin S10b varied with individual tumors tested. The response rate of liblomycin, a myelotoxic compound within this normalized range, appears promising. These data represent the first comparison of liblomycin to bleomycin against a spectrum of fresh human tumors using a stem cell assay technique.

In vivo mineral contents of dietary fiber determined by EDX analysis.

EDX measurements of the mineral content of oat hulls in the pig GI tract revealed that the hulls from the rectum contained more Na, P and S than prior to ingestion; while contents of K and Ca were not significantly different than in initial hulls. X-ray measurements indicated that potassium was the only element of the five examined which was unloaded from the initial oat hulls during passage through the GI tract. During this passage, the flows of Na and K into and out of oat hulls were proportional to concentrations of these elements in digesta. Although Ca was loaded onto oat hulls in the stomach, it was unloaded during subsequent passage, even though the concentration of Ca in digesta increased significantly in the large intestine. The behavior of corn pericarp generally was similar to that of oat hulls. We consider it unwise to attempt quantitative comparisons of corn pericarp behavior in our previous work (Dintzis et al., in press) with results in this study. The data bases are very small and feeding situations and diets were not equivalent. However, results from both studies show similar behavior in loading and unloading of Na, Ca, and S from the plant tissues, and possible differences with K and P. The finding that X-ray count values were different for oat hulls and corn pericarp when the tissues were loaded with equal amounts of calcium or potassium serves as a stark warning that additional work is required before measurements of this sort can provide quantitative results and a basis for comparisons between different plant tissues. Nevertheless, EDX analysis has provided information about the loading of Na, K, Ca, S, and P onto plant tissues during passage in the GI tract of growing pigs. The techniques used in this study appear worthy of further effort, for we believe they have the potential to provide significant information about in vivo interactions between plant tissues and minerals in the diet.

Energy-dispersive X-ray analysis of the mineral content of corn bran treated in vitro and by passage through the pig gastrointestinal tract.

Energy-dispersive X-ray (EDX) analysis was tested as a method for examining the mineral contents of corn bran loaded in vitro or passed through the GI tract of pigs. Particles of dry-milled corn pericarp treated in vitro or retrieved from the stomach, ileum, and colon of killed pigs were prepared as microtomed bulk specimens directly embedded in resin. Because of heterogeneity caused by differences in substrate cell density and mineral content, X-ray count averages for a number of different specimens had a coefficient of variation greater than or equal to 0.24. Detectable amounts of K, Ca, S, and P, but not Na, Cu, Fe, or Zn were found in specimens of initial bran. Although detectable concentrations of Cu, Fe and Zn could be loaded in vitro, these elements generally were not detected in bran retrieved from the pig GI tract. During GI tract passage, sodium was loaded onto bran mainly in the small intestine and unloaded in the large intestine. Calcium was loaded in the stomach and unloaded mainly in the small intestine. At each GI tract location, content of Na, Ca, K, and P in retrieved bran was greater than in the initial bran. EDX microprobe methods can be applied successfully to plant tissues treated in vitro and in vivo to investigate interactions with minerals in a diet.

Antitumor activity against human tumor samples of cis-diamminedichloroplatinum(II) and analogues at equivalent in vitro myelotoxic concentrations.

We compared the antitumor activity of cis-diamminedichloroplatinum(II) (cisplatin; CDDP) with three CDDP analogues: cis-diammine-1,1-cyclobutanedicarboxylateplatinum(II) (CBDCA), N-methyliminodiacetato-1,2-diamino(cyclohexane)platinum(II) (MIDP), and N-(2-hydroxyethyl)-iminodiacetato-1,2-diamino(cyclohexane)platinum (II) (HIDP). Fresh human tumor samples in the adhesive tumor culture system were utilized for this comparison. The equitoxic concentrations of all four drugs were derived based on their inhibitory activity against human bone marrow samples. For these normalized concentrations, CDDP proved to have a higher cytotoxic activity than its analogues. CBDCA's in vitro activity had a significant correlation with CDDP activity (r = 0.67) in vitro. However, the structurally similar substances MIDP and HIDP demonstrated a much greater degree of association (r = 0.90). Our data suggest that CBDCA, HIDP, and MIDP have overall less activity than CDDP when tested at equitoxic in vitro concentrations. Close association between CDDP and CBDCA also reflects known clinical experience with these two drugs, suggesting the method of comparison used here is probably appropriate. These conclusions, however, must be validated by clinical trials.

In vitro activity of amonafide against primary human tumors compared with the activity of standard agents.

Amonafide, one of a series of benz[de]-isoquinoline-1,3-dione compounds, is now entering phase II clinical trials in this country. We tested amonafide, exposed continuously for 5 days, at four different concentrations against 56 primary human tumors in vitro. The drug concentration range used was based on amonafide's inhibitory activity against human bone marrow cells. The antitumor activity of 5-fluorouracil, mitomycin C, cisplatin, and etoposide against tumors from this panel of 56 was compared with that of amonafide at in vitro concentrations equitoxic against human bone marrow cells. Amonafide was active against only 12% of tumors compared with standard agents, which were active against more than 40% of tumors in the human bone marrow inhibitory range. Our data suggested that amonafide is less likely to be clinically active against human solid tumors than the standard agents.

A microfibril-generating factor from the cellulase of Trichoderma reesei.

A combination of ionic strength reduction and diafiltration of Trichoderma reesei cellulate complex through a hollow fiber apparatus of 5000 molecular weight (MW) cutoff and subsequent passage of filtrate over a Spherogel-TSK 3000-SW column provided extracts that had the ability to generate microfibrils in filter paper and to disrupt filter paper and corn leaf tissue. Milligram quantities of material obtained from these extracts released small amounts of soluble carbohydrate from filter paper, required ferric iron for increased activity, and contained amino acids. Short fiber formation and disruption of filter paper during interaction with these extracts was enhanced by prior acid treatment and eliminated by prior base treatment. The amount of soluble carbohydrate hydrolyzed in 24 h from filter paper by whole cellulase complex was not changed by first disrupting the substrate with the extracts.

High colony-forming efficiency of primary human tumor cells cultured in the adhesive-tumor-cell culture system: improvements with medium and serum alterations.

The colony-forming efficiency (CFE) of primary human tumor cells cultured in the adhesive-tumor-cell culture system (ATCCS) using Ham's F12 (F12) or Eagle's minimum essential medium, alpha modification (alphaMEM) and culture medium supplemented with either swine, equine or bovine sera were compared. AlphaMEM supplemented with equine serum provided the highest CFE of the combinations. The CFE increase due to the change from F12 to alphaMEM was approximately 5-fold, and the increase due to the change in serum from swine to equine was approximately 2-fold. Cytokeratin staining showed that this increase was not due to fibroblast growth. The high-average CFE with alphaMEM, approximately 3%, means that an inoculum of only 2 X 10(3) cells is needed to achieve formation of approximately 65 colonies in control cultures, thereby increasing the performance of this system when used in a chemosensitivity assay.

Drug and radiation sensitivity testing of primary human tumor cells using the adhesive-tumor-cell culture system (ATCCS).

In summary, the ATCCS is an efficient culture system which grows clonogenic cells from greater than 80% of human cancers. The ATCCS supports the growth of malignant cells from human cancers. The ATCCS shows classical drug and radiation survival curves which routinely achieve over 1 log of kill. The ATCCS has high CFE and permits sensitivity testing from small samples. Clinical correlations for the chemosensitivity assay were satisfactory. Radiosensitivity in vitro correlates with tumor histology.

Involvement of chromosome 7 in primary lung tumor and nonmalignant normal lung tissue.

By using the newly developed adhesive tumor cell culture system, we analyzed the chromosomal constitutions of primary lung tumor and nonmalignant normal lung tissue from 10 previously untreated patients with non-small cell lung cancer. Chromosomal analyses were successfully carried out in banded chromosome preparations from 10 tumor and 8 normal lung tissue samples. All analyzed tumor and normal lung tissue samples had a predominantly normal diploid chromosome number. However, there was at least one structural or numerical alteration in every tumor and lung tissue sample analyzed. Chromosomes 1, 3, 4, 6, 7, 8, 9, 12, 15, and 20 were more often involved in rearrangement. The most consistent finding was trisomy 7; 4 patients had trisomy 7 in both tumor and normal lung tissue, and another 2 had this anomaly in tumor tissue only. Of the 4 patients without trisomy 7, 2 had a homogeneously staining region in the short arm of chromosome 7 in tumor tissue. Phytohemagglutinin-stimulated peripheral blood lymphocytes from 7 patients, including 5 patients with trisomy 7 in tumor tissue, did not show trisomy 7. These cytogenetic data suggest that chromosome 7 may be associated with lung cancer development and that trisomy 7 may be the hallmark of premalignant changes, at least in a subgroup of patients with non-small cell lung cancer.

Comparison between clinical response and in vitro drug sensitivity of primary human tumors in the adhesive tumor cell culture system.

The newly described adhesive tumor cell culture system (ATCCS) offers a distinct advantage over other assays in that it has a high plating efficiency requiring low cell inoculum, it affords workable assays in approximately 70% of specimens from the heterogenous tumor types, and it has the ability to assay up to nine drugs at four different concentrations. Clinical correlations based on the ATCCS were obtained in 65 patients undergoing 71 clinical trials. Patients with melanoma, lung cancer, and sarcoma dominated the group. The most active in vitro drug was correlated per clinical trial. Thirteen of 17 (76%) sensitive in vitro predictions and 51 of 54 (94%) resistant in vitro predictions were accurate. The assay in this study had a sensitivity of 81% and specificity of 93%. These preliminary results are encouraging and warrant prospective trials to establish the true value of this assay to patients.

Comparison of antitumor activity of standard and investigational drugs at equivalent granulocyte-macrophage colony-forming cell inhibitory concentrations in the adhesive tumor cell culture system: an in vitro method of screening new drugs.

We compared the in vitro growth inhibition of primary human tumor cells in the adhesive tumor cell culture system (ATCCS), exposed to the investigational agents caracemide, spirogermanium and taxol and to standard chemotherapy agents at equitoxic concentrations for granulocyte-macrophage colony-forming cells (GM-CFC) in vitro. Clinically active standard agents tested at up to GM-CFC 90% inhibitory concentrations (IC90) resulted in in vitro activity (greater than or equal to 50% tumor growth inhibition) in at least 30% of tumors tested. In vitro responses for taxol, caracemide and spirogermanium were 78%, 9% and 7%, respectively. This paper proposes a model that incorporates two hypotheses: (1) myelotoxic drugs which inhibit tumor growth at concentrations equal to or less than equitoxic GM-CFC ICs will demonstrate clinical activity; and (2) both myelotoxic and particular nonmyelotoxic drugs inactive in vitro at these doses will not be active clinically. If this drug screening concept is valid, taxol may be clinically more active than caracemide and spirogermanium.

Biological effect of epidermal growth factor on the in vitro growth of human tumors.

The effect of epidermal growth factor (EGF) on the in vitro growth of 186 malignant human tumor specimens (45 melanomas, 32 sarcomas, and 56 lung, 16 gynecological, 14 breast, 12 genitourinary, and 11 gastrointestinal carcinomas) was evaluated in the cellular adhesive matrix human tumor culture system supplemented with transferrin, insulin, hydrocortisone, and estradiol. EGF increased tumor growth by at least 50% in 81% of the 186 tumors and by over 100% in 54%. The enhanced growth induced by EGF was related to an accelerated cellular division independent of tumor type and not to an increase in the actual number of clonogenic units. The drug concentrations of cell cycle-independent Adriamycin and cisplatin needed to achieve a 90% tumor cell kill were not altered by the responsiveness of the tumor to EGF.