Role of alloantigens in natural killing. Allogeneic but not autologous tumor biopsy cells are sensitive for interferon-induced cytotoxicity of human blood lymphcoytes.

Blood lymphocytes of patients with solid tumors were assayed for cytotoxicity against autologous and allogeneic primary tumor cells. The lymphocytes killed autologous tumor cells in 7 of 25 cases (28%) and allogeneic tumor cells in 2 of 37 tests (5%). Lymphocytes from healthy donors were rarely cytotoxic for the biopsy cells, which indicates that these cells have low natural kill sensitivity. The autoreactivity that may reflect the immunological recognition of tumor cells was not altered by pretreatment of the effectors with interferon (IF). In contrast, killing of allogeneic tumor biopsy cells was induced by IF in approximately 50% of tests, with the lymphocytes of both the tumor patients and the healthy donors. The mechanism of the alloreactivity is most likely a consequence of IF-induced polyclonal activation of cytotoxic potential and the lymphocytes that are committed to recognize the alloantigens expressed on the particular target manifest the killing function. When the biopsy cells were explanted and kept in culture for 5-6 d, their susceptibility for the lymphocyte damage increased, and they were killed by the IF-treated cells also in autologous combinations. Whether this change in sensitivity is a result of qualitative or quantitative changes in antigen expression or of other changes in the properties of the cell membrane is unknown.

Induction of 2',5'-oligoadenylate synthetase and blast transformation in primary chronic lymphocytic leukemia cells following exposure to interferon in vitro.

Interferon (IFN) can induce blast transformation and differentiation of malignant cells from patients with chronic lymphocytic leukemia (CLL). In this work the capacity of IFN to induce 2',5'-oligoadenylate synthetase (2',5'-A synthetase) in lymphoid cells from patients with CLL was investigated, and the results were related to the induction of blast transformation. IFN induced enhanced levels of 2',5'-A synthetase in unseparated lymphoid cells from 18 of 24 patients with CLL. In a control group of 11 healthy donors, 2',5'-A synthetase was induced in all cases tested. There was a close correlation between induction of 2',5'-A synthetase and induction of blast transformation by IFN. Thus, transformation occurred in clones expressing enhanced levels of 2',5'-A synthetase, but not in those showing no increase in 2',5'-A synthetase. An enhancement of 2',5'-A synthetase was observed in the IFN-sensitive cells following exposure to concentrations as low as 0.5 IFN units/ml. For induction of blast transformation, 10-1000 times more IFN was required. One h of pretreatment was sufficient for induction of 2',5'-A synthetase, whereas 20 h of pretreatment were required for induction of transformation by IFN. The finding that induction of 2',5'-A synthetase parallels interferon-induced blast transformation indicates that the reason why some CLL clones do not differentiate following exposure to IFN is a resistance of these cells to the action of IFN. The resistance to IFN in some CLL clones may be due to a defect in the 2',5'-A synthetase system of the cells, but it could also be at an early stage of the interaction between IFN and the cell, for instance at the receptor level.

Deletion of alpha-, beta-, and omega-interferon genes in malignant cells from children with acute lymphocytic leukemia.

Scanning densitometry and restriction fragment length polymorphism analysis were used to study the alpha-, beta-, gamma-, and omega-interferon (IFN) genes in malignant cells from 11 children with acute lymphocytic leukemia and in one cell line of T-cell origin. In the malignant cells of one patient there was a complete loss of alpha-, beta-, and omega-IFN genes, whereas in another patient one of the alleles of these genes had been deleted. Cytogenetic analysis revealed a deletion of the short arm of chromosome 9, i.e., the region containing the alpha-, beta-, and omega-IFN genes, in the latter patient. The normal cells of the patients with IFN gene deletions had two alleles of the alpha-, beta-, and omega-IFN genes. In cells from none of the patients could deletions or rearrangements of the gamma-IFN genes be detected. We conclude that in 2 of 11 patients with acute lymphocytic leukemia the malignant transformation is accompanied by loss of material on one or both chromosomes 9 and that the alpha-, beta-, and omega-IFN genes are included in these deletions.

Alpha-interferon receptors in malignant B-cells from patients with chronic lymphocytic leukemia: relation to induction of 2'-5'-oligoadenylate synthetase and blast transformation.

alpha-Interferon (IFN-alpha) induces blast transformation of malignant B-cells from approximately 65% of chronic lymphocytic leukemia patients. We have shown previously that induction of blast transformation correlates with induction of 2'-5'-oligoadenylate synthetase. In this paper we address the question of whether low responsiveness to IFN-alpha is associated with a reduced expression of the IFN receptor. IFN-alpha receptor expression was studied by the binding of radioiodinated IFN-alpha to peripheral blood malignant B-cells from 20 chronic lymphocytic leukemia patients and to blood cells from 5 healthy donors. Chronic lymphocytic leukemia cells from all 20 patients displayed high affinity IFN-alpha receptors [mean Kd, 62 +/- 9 (SE) pM] ranging between 110 and 850 binding sites/cell [mean, 416 +/- 51]. Nonmalignant mononuclear blood cells showed similar binding data (411 +/- 105 binding sites/cell; Kd 66 +/- 20 pM). Receptor expression did not correlate with the degree of blast transformation or with induction of 2'-5'-oligoadenylate synthetase. We conclude that the deficiency of IFN sensitivity is localized somewhere between signal transduction from the receptor and induction of 2'-5'-oligoadenylate synthetase.

Induction of Cip/Kip and Ink4 cyclin dependent kinase inhibitors by interferon-alpha in hematopoietic cell lines.

One prominent effect of IFNs is their cell growth inhibitory activity. The exact molecular mechanism behind this inhibition of proliferation remains to be elucidated. Possible effectors for IFN-induced growth inhibition are the recently discovered cyclin-dependent kinase inhibitors. The effect of IFN-alpha treatment on the members of the Ink4 and Cip/Kip families of Cdk inhibitors was investigated in three hematopoietic cell lines Daudi, U-266 and H9. Two of these cell lines, Daudi and U-266, respond to IFN-alpha by G1 arrest, whereas the H9 cell line is not growth arrested by IFN-alpha. We show that a p53-independent upregulation of p21 mRNA occurs following IFN-alpha treatment in all three cell lines. In Daudi and U-266 cells, the mRNA induction is accompanied by an increase in p21 protein, followed by an increased binding of p21 to Cdk2 and a subsequent decrease in Cdk2 activity, temporally coinciding with G1 arrest. In both these cell lines, there was also an increased binding of p21 to Cdk4. In contrast, p21 protein was not expressed in H9 cells, despite high levels of p21 mRNA following IFN-alpha treatment. In U-266 cells, IFN-alpha increased not only p21 but also p15 mRNA and protein levels, followed by an increased association of p15 with Cdk4. Furthermore, IFN-alpha treatment caused a four- to sixfold induction of the p16 E1beta transcript in U-266 cells. Expression levels of the other Ink4 and Cip/Kip Cdk inhibitors were not induced by IFN treatment in any of the cell lines. We conclude that IFN-alpha can act as a potent regulator of Cdk-inhibitor expression, correlating with decreased Cdk activity and cell growth inhibition. One mechanism for resistance to IFN may be loss of the ability of cells to upregulate these proteins.

Involvement of the Ink4 proteins p16 and p15 in T-lymphocyte senescence.

Little is known about the molecular background to senescence in T-lymphocytes. In fibroblast systems replicative senescence has been shown to correlate with a number of changes in the expression of the proteins normally regulating progression through the G1 phase of the cell cycle, and recently the Ink4 inhibitor p16 was implicated as a central regulator of replicative senescence in human fibroblasts. It has, however, been claimed that p16 is not expressed in T-lymphocytes. In the present study we have analysed G1 regulating proteins in ageing human T-lymphocytes. We show that PHA and IL-2 stimulated T-lymphocytes cease to proliferate after around 20 population doublings, these cells can not thereafter be restimulated to growth, and were also found to exhibit markers for senescence. We found that T-lymphocytes accumulate p16 and p15 protein during successive population doublings and display high levels of these proteins as they enter into replicative senescence. There was also an increased binding of p16 to the Cdk6 kinase in senescent cells, and a decreased Cdk6 as well as Cdk2 kinase activity. The levels of other G1 regulating proteins were also altered in the senescent cells, such as slightly elevated levels of p21/WAF1, and downregulation of Cdk2 and cyclinD3. The levels of p27/ Kip1 is down regulated in proliferating cells but rise to approximately 15% of the levels in un-stimulated quiescent cells. As a high proportion of T-cell childhood acute lymphoblastic leukaemias have deletions of both p15 and p16, our data suggest that inactivation of these genes makes it possible for leukemic cells to avoid senescence.

Molecular mechanisms underlying interferon-alpha-induced G0/G1 arrest: CKI-mediated regulation of G1 Cdk-complexes and activation of pocket proteins.

One prominent effect of IFNs is their cell growth-inhibitory activity. The mechanism behind this inhibition of proliferation is still not fully understood. In this study, the effect of IFN-alpha treatment on cell cycle progression has been analysed in three lymphoid cell lines, Daudi, U-266 and H9. Examination of the growth-arrested cell populations shows that Daudi cells accumulate in a G0-like state, whereas U-266 cells arrest later in G1. H9 cells are completely resistant to IFN-alpha's cell growth-inhibitory effects. The G0/G1-phase arrest is preceded by a rapid induction of the cyclin-dependent kinase inhibitors (CKIs), p21 and p15. In parallel, the activities of the G1 Cdks are significantly reduced. In addition to p21/p15 induction, IFN-alpha regulates the expression of another CKI, p27, presumably by a post-transcriptional mechanism. In the G1 Cdk-complexes, there is first an increased binding of p21 and p15 to their respective kinases. At longer exposure times, when Cdk-bound p15 and p21 decline, p27 starts to accumulate. Furthermore, we found that IFN-alpha not only suppresses the phosphorylation of pRb, but also alters the phosphorylation and expression of the other pocket proteins p130 and p107. These data suggest that induction of p21/p15 is involved in the primary IFN-alpha response inhibiting G1 Cdk activity, whereas increased p27 expression is part of a second set of events which keep these Cdks in their inactive form. Moreover, elevated levels of p27 correlated with a dissociation of cyclin E/Cdk2-p130 or p107 complexes to yield cyclin E/Cdk2-p27 complexes. In resistant H9 cells, which possess a homozygous deletion of the p15/p16 genes and lack p21 protein expression, IFN-alpha causes no detectable changes in p27 expression and, furthermore, no effects are observed on either pocket proteins in this cell line. Taken together, these data suggest that the early decline in G1 Cdk activity, subsequent changes in phosphorylation of pocket proteins, and G1/G0 arrest following IFN-alpha treatment, is not primarily due to loss of the G1 kinase components, but result from the inhibitory action of CKIs on these complexes.

Cloning of two candidate tumor suppressor genes within a 10 kb region on chromosome 13q14, frequently deleted in chronic lymphocytic leukemia.

Previous studies have indicated the presence of a putative tumor suppressor gene on chromosome 13q14, commonly deleted in patients with B-cell chronic lymphocytic leukemia (B-CLL). We have previously defined a minimally deleted region of 130 kb centromeric to the marker D13S272, and constructed a PAC and cosmid contig encompassing this area. In the present study we have made a detailed restriction and transcriptional map of the region of interest. Using these tools we have screened a panel of 206 primary CLL clones and three cell lines. In five CLL cases we found limited deletions defining the region of interest to an area of no more than 10 kb. Two adjacent genes, termed Leu1 and Leu2 (leukemia-associated gene 1 and 2), were mapped to the minimally deleted region, with several patients showing deletion borders within these genes. The Leu1 and Leu2 genes show little homology to previously published genes at the nucleotide and expected translated amino acid sequence level. Mutational analysis of the Leu1 and 2 genes in 170 CLL samples revealed no small intragenic mutations or point mutations. However, in all cases of 13q14 loss examined, the first exon of both genes, which are only 300 bp apart, were deleted. We conclude that the Leu1 and Leu2 genes are strong candidates as tumor suppressor gene(s) involved in B-CLL leukemogenesis.

Prognostic importance of p15INK4B and p16INK4 gene inactivation in childhood acute lymphocytic leukemia.

PURPOSE: The present study explores the prognostic importance of p16INK4/p15INK4B gene inactivation in childhood acute lymphocytic leukemia (ALL). MATERIALS AND METHODS: Cells from 79 pediatric ALL patients were investigated for inactivation of the p15INK4B and p16INK4 genes or loss of heterozygosity (LOH) for chromosome 9p markers by use of Southern hybridization, restriction fragment length polymorphism (RFLP) analysis, microsatellite analysis as well as single-strand conformation polymorphism (SSCP) analysis, and nucleotide sequencing of the p15INK4B and p16INK4 genes. Genetic data were correlated to clinical outcome and established prognostic factors. RESULTS: Inactivation of the p15INK4B and/or p16INK4 genes by homozygous deletion or loss of one allele and mutation of the other was detected in 24 cases (30%). Another 12 patients (15%) showed loss of one allele. A statistically significant correlation was found between inactivation of the p15INK4B/p16INK4 genes and poor prognosis (P < .01). Furthermore, inactivation proved to be an independent factor that predicted relapse, ranking second to WBC count. The trend toward overrepresentation of treatment failure was strongest in the high-risk (HR) group patients with p16INK4/p15INK4B gene inactivation. Patients with deletion of genetic material on 9p21 and normal coding sequence of the remaining p16INK4 and p15INK4B genes had a similar prognosis to that of nondeleted cases. CONCLUSION: The data suggest that analysis of p15INK4B/p16INK4 genes may contribute prognostic information in pediatric ALL.

Gamma-IFN induced cell adhesion in chronic myelogenous leukemia cells.

Both gamma- and alpha-interferons (IFNs) have been shown to induce clinical responses in chronic myelogenous leukemia (CML). The mechanisms behind these effects are not known. In chronic phase CML, granulocytic progenitors normally found in the bone marrow only, are found extramedullary. A defect in the adhesion of CML cells, that may be responsible for this finding, has been described earlier. In this study, we have investigated whether IFN can restore the defect in CML cell adhesion. It was found that gamma-, but not alpha-IFN, strongly induced the adhesion of CML cells to other cells and to plastic in a majority of the patients. In parallel, an induction in the expression of the adhesion molecules ICAM-1 and LFA-1 was found, and blocking of these molecules by antibodies abolished the effect. The ability of gamma-IFN to restore the adhesive property of CML cells may add to the antitumor effects observed with gamma-IFN therapy in CML.

Molecular analyses of chromosome 12 in chronic lymphocytic leukemia.

Trisomy 12 is the most common chromosomal aberration in chronic B lymphocytic leukemia (B-CLL). In this study we have investigated trisomy 12 and posed two major questions: (a) What is the origin of the third copy of chromosome 12? and (b) What is the proportion of trisomy 12 cells in malignant clones with this aberration? The origin of an extra copy of chromosome 12 in lymphocytes from patients with B-CLL was studied by the use of probes detecting restriction fragment length polymorphisms on this chromosome. In all six patients that were evaluable, the third copy was derived from a simple duplication of one of the original chromosomes. In none of these patients nor in four patients with two copies of chromosome 12 were losses of the homologue observed. When studying metaphase cells from some CLL patients with trisomy 12, a large proportion of the cells are found to have a normal karyotype. In this study the fraction of normal metaphases was not matched by a similar fraction of cells lacking trisomy 12, as judged by scanning densitometry of hybridization bands. Thus, normal metaphases appear to be derived from a small fraction of easily stimulated probably nonmalignant cells and not from a large second population of malignant cells with a normal karyotype.

Interferon system defects in malignant T-cells.

Deletions of chromosome 9p21-22 occur in acute lymphocytic leukemia (ALL), melanoma and glioma. With some exceptions, these deletions include the alpha- and beta-interferon (IFN) genes. In this study, the frequency of alpha- and beta-IFN gene deletions was investigated in 17 T-cell lines, and losses of IFN genes were related to other aspects of the IFN system. Deletions of alpha-/beta-IFN genes were observed in 7/17 cell lines. In two cases the deletions were homozygous for both loci. In most cases aberrations of chromosome 9 were also apparent on cytogenetic analysis. An increased proportion (40% as compared to the expected 13%) of the remaining ten cell lines showed homozygosity for all five common polymorphic alpha-/beta-IFN markers, possibly implicating allelic deletion by loss of heterozygosity (LOH) in some of these clones. The cell lines showed a large variability in IFN production, IFN-alpha receptor number, susceptibility to IFN measured as induction of the enzyme 2',5' oligoadenylate synthetase and cell growth inhibition. No correlations between loss of IFN genes and IFN-producing capacity, or susceptibility to IFN, were found. Of the seven cell lines with a normal IFN-gene dosage and heterozygosity for the alpha- and beta-IFN genes, three had a deficiency in their IFN-producing capacity and one was also insensitive to growth inhibition by IFN. All IFN-producing cell lines predominantly produced beta-IFN.

A FISH cosmid 'cocktail' for detection of 13q deletions in chronic lymphocytic leukaemia--comparison with cytogenetics and Southern hybridization.

The most frequent structural chromosome abnormality in chronic lymphocytic leukaemia (CLL) is deletion at chromosome 13q14. Studies with Southern blot hybridisation have revealed deletions in the region located telomeric of the retinoblastoma gene in more than 40% of cases. The highest frequency of homozygous deletions has been found at the D13S319 locus and it is likely that a new tumour suppressor gene is located close to this region. We have analysed deletions in the D13S319 region in 20 selected CLL patients using conventional cytogenetic analysis, fluorescence in situ hybridisation (FISH) and Southern blot hybridisation. FISH and Southern hybridisation are equally efficient in detecting deleted clones in our study. However, FISH analysis indicate that subclones with different numbers of alleles in the D13S319 region can exist simultaneously. The cytogenetic analyses confirm that clones with different chromosomal abnormalities can occur in patients with CLL and that 13q14 deletions can be limited to one of these subclones. Furthermore, the FISH analyses show that trisomy 12 and deletion of 13q14 can occur in the same cell clone. Finally, our study confirms that mitogen stimulation of peripheral blood cells from CLL patients before FISH analysis may result in a sharp increase in normal appearing cells, which can hide leukaemic clones with deletions in the D13S319 region.

Deletion of the Ink4-locus (the p16ink4a, p14ARF and p15ink4b genes) predicts relapse in children with ALL treated according to the Nordic protocols NOPHO-86 and NOPHO-92.

Inactivation of the Ink4 gene locus locus on 9p comprising the tumour suppressor gene p16ink4a and its neighbours p14ARF and p15ink4b is common in childhood acute lymphoblastic leukaemia (ALL), but the prognostic significance is controversial. DNA from 230 patients was retrospectively analysed by Southern blotting, single strand conformation polymorphism (SSCP) and sequencing techniques. The results were correlated with clinical characteristics and outcome. One hundred and ninety-four fully analysed patients, similarly treated using the Nordic NOPHO-86 or the current NOPHO-92 protocols, were included in the outcome analysis. Deletions approached a minimally deleted region between the p16ink4a and p15ink4b genes, making the p14ARF gene the most commonly deleted coding sequence. Bi-allelic deletion was associated with high white blood cell count (WBC) (P < 0.001), T cell phenotype (P < 0.001) and mediastinal mass (P < 0.001). Patients with Ink4 locus bi-allelic deletions had an inferior pEFS (P < 0.01) and multivariate analysis indicated that bi-allelic deletion of the p16ink4a and the p14ARF genes was an independent prognostic risk factor (P < 0.05). Sub-group analysis revealed a pronounced impact of deletion status for high-risk patients, ie with high WBC. Deletion-status and clinical risk criteria (WBC) could thus be combined to further differentiate risk within the high-risk group. The analysis of the Ink4 locus adds independent prognostic information in childhood ALL treated by Nordic protocols and may help in selection of patients for alternative treatment.

Transitory hypocalcemia complicating measles.

During an epidemic of measles, 12 young adults developed hypocalcemia associated with a decrease in parathyroid hormone level. Calcium and parathyroid hormone levels returned to normal in five of the 12 patients for whom convalescent data were available.

Combinations of interferon-gamma and retinoic acid or 1 alpha, 25-dihydroxycholecalciferol induce differentiation of the human monoblast leukemia cell line U-937.

The monoblastlike leukemia cell line, U-937, is induced to differentiate into monocytelike cells by incubation with 200-500 U/ml of recombinant human immune interferon (IFN-gamma) judging from capacity to reduce nitroblue tetrazolium. At least an additive differentiation-inducing effect was found between IFN-gamma and 1-100 nM retinoic acid (RA). A marked synergistic differentiation-inducing effect was found between IFN-gamma and 0.1-1.0 nM 1 alpha,25-dihydroxycholecalciferol (1,25[OH]2D3). It is also shown that U-937 can be primed for differentiation by treatment for approximately one day with 1,25(OH)2D3 followed by exposure to IFN-gamma. Priming of these cells does not depend on the normal rate of RNA synthesis, as it occurs even better in the presence of cordycepin, suggesting that a decrease in RNA synthesis favors IFN-induced differentiation. Actually, the addition of cordycepin during initial incubation with IFN increased the subsequent response to IFN-gamma (and also to RA and 1,25[OH]2D3). These results, indicating that combinations of IFN-gamma and either RA or 1,25(OH)2D3 induce differentiation of U-937, may be of importance in combination biotherapy of leukemia.

A cosmid and cDNA fine physical map of a human chromosome 13q14 region frequently lost in B-cell chronic lymphocytic leukemia and identification of a new putative tumor suppressor gene, Leu5.

B-cell chronic lymphocytic leukemia (B-CLL) is a human hematological neoplastic disease often associated with the loss of a chromosome 13 region between RB1 gene and locus D13S25. A new tumor suppressor gene (TSG) may be located in the region. A cosmid contig has been constructed between the loci D13S1168 (WI9598) and D13S25 (H2-42), which corresponds to the minimal region shared by B-CLL associated deletions. The contig includes more than 200 LANL and ICRF cosmid clones covering 620 kb. Three cDNAs likely corresponding to three different genes have been found in the minimally deleted region, sequenced and mapped against the contigged cosmids. cDNA clone 10k4 as well as a chimeric clone 13g3, codes for a zinc-finger domain of the RING type and shares homology to some known genes involved in tumorigenesis (RET finger protein, BRCA1) and embryogenesis (MID1). We have termed the gene corresponding to 10k4/13g3 clones LEU5. This is the first gene with homology to known TSGs which has been found in the region of B-CLL rearrangements.

Why do so many cancer patients fail to respond to interferon therapy?

Since their first use in the clinic some 25 years ago, interferons (IFNs) have become accepted therapy in a range of cancer forms. However, although in some patients they induce remission, in the great majority they are of no benefit or, at best, lead only to minor improvements. This review considers possible reasons for these failures.

Cytotoxic effect of interferon on primary malignant tumour cells. Studies in various malignancies.

It is a well established fact that interferon (IFN) can inhibit cell growth, but only recently has it been found that IFN can exert a direct cytotoxic effect on primary tumour cells. This was shown in malignant cells from patients with multiple myeloma. In this study the influence of IFN on the viability of primary malignant cells from patients with different malignancies was studied. As previously described a direct cytotoxic effect of IFN on multiple myeloma cells was observed. No major effects on cell viability could be found in malignant cells from patients with lymphoma, chronic lymphocytic leukaemia, hairy cell leukaemia, chronic myelogenous leukaemia and carcinoma. This indicates that the direct cytotoxic effect of IFN in multiple myeloma may be relatively specific for this malignancy. It could be due to a specific differentiation stage in the myeloma cells, specific genetic alterations and/or abrogation of an autocrine/paracrine loop.

Measurement of interferon sensitivity in tumour cells from fine-needle aspirations.

Previous studies have shown significant correlations between interferon (IFN) induced enhancement of the enzyme 2',5'-oligoadenylate (2',5'-A) synthetase in vitro and response to IFN therapy. A limitation of this and other predictive tests is the availability of malignant cells for culture. Malignant cells can be obtained from most palpable solid tumours by fine-needle aspiration. We investigated whether malignant cells from such aspirations can be used in a 2',5'-A synthetase assay. In 23/27 (85%) of the cases sufficient amounts of viable cells were obtained, containing a high proportion (greater than or equal to 90%) of tumour cells. In 13/23 tumour samples (57%) IFN-alpha significantly enhanced the 2',5'-A synthetase levels. The use of cells from the fine-needle aspirations for prediction of IFN sensitivity, makes the 2',5'-A synthetase test applicable in a wide range of tumours at a variety of disease stages.