Steel factor sustains SCL expression and the survival of purified CD34+ bone marrow cells in the absence of detectable cell differentiation.

CD34+ cells express the basic helix-loop-helix transcription factor SCL, which is essential for blood cell formation in vivo. In addition, their survival is critically dependent on hemopoietic growth factors. We therefore compared the effects of Steel factor (SF) and GM-CSF on the survival, proliferation, and differentiation of primary human CD34+ cells, as well as the role of SCL during these processes. GM-CSF suppresses apoptosis in CD34+ cells, which proliferate and differentiate into mature granulocytic and monocytic cells (CD34-CD13+) and loose SCL expression. In contrast, SF suppresses apoptosis without a significant increase in cell numbers, and the cells remain CD34+ and SCL+ with a blast-like morphology. Examination of apoptosis by the terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL) reaction and of the cell cycle status indicated that SF is both a survival factor and a mitogenic factor for CD34+ cells. There was, however, constant cell death in a fraction of the population, which could be rescued by GM-CSF. Co-addition of SF and GM-CSF prevents the downregulation of SCL observed in the presence of GM-CSF by itself, allows for prolonged survival and expansion of CD34+ cells in culture, inhibits monocytic differentiation and impairs granulocytic differentiation. Finally, exposure to an antisense SCL but not a control oligonucleotide decreases SCL protein levels and prevents the suppression of apoptosis by SF without affecting GM-CSF-dependent cell survival. These observations suggest that the hemopoietic transcription factor SCL regulates the survival of CD34+ cells in response to SF.

Transcription factor SCL is required for c-kit expression and c-Kit function in hemopoietic cells.

In normal hemopoietic cells that are dependent on specific growth factors for cell survival, the expression of the basic helix-loop-helix transcription factor SCL/Tal1 correlates with that of c-Kit, the receptor for Steel factor (SF) or stem cell factor. To address the possibility that SCL may function upstream of c-kit, we sought to modulate endogenous SCL function in the CD34(+) hemopoietic cell line TF-1, which requires SF, granulocyte/macrophage colony-stimulating factor, or interleukin 3 for survival. Ectopic expression of an antisense SCL cDNA (as-SCL) or a dominant negative SCL (dn-SCL) in these cells impaired SCL DNA binding activity, and prevented the suppression of apoptosis by SF only, indicating that SCL is required for c-Kit-dependent cell survival. Consistent with the lack of response to SF, the level of c-kit mRNA and c-Kit protein was significantly and specifically reduced in as-SCL- or dn-SCL- expressing cells. c-kit mRNA, c-kit promoter activity, and the response to SF were rescued by SCL overexpression in the antisense or dn-SCL transfectants. Furthermore, ectopic c-kit expression in as-SCL transfectants is sufficient to restore cell survival in response to SF. Finally, enforced SCL in the pro-B cell line Ba/F3, which is both SCL and c-kit negative is sufficient to induce c-Kit and SF responsiveness. Together, these results indicate that c-kit, a gene that is essential for the survival of primitive hemopoietic cells, is a downstream target of the transcription factor SCL.

Cellular proliferation and transformation induced by HOXB4 and HOXB3 proteins involves cooperation with PBX1.

The products of PBX homeobox genes, which were initially discovered in reciprocal translocations occurring in human leukemias, have been shown to cooperate in the in vitro DNA binding with HOX proteins. Despite the growing body of data implicating Hox genes in the development of various cancers, little is known about the role of HOX-PBX interactions in the regulation of proliferation and induction of transformation of mammalian cells. We build on the existing model of Hox-induced transformation of Rat-1 cells to show that both cellular transformation and proliferation induced by Hoxb4 and Hoxb3 are greatly modulated by the levels of available PBX1 present in these cells. Furthermore, we show that the transforming capacity of these two HOX proteins depends on their conserved tetrapeptide and homeodomain regions which mediate binding to PBX and DNA, respectively. Taken together, results of this study demonstrate that cooperation between HOX and PBX proteins modulates cellular proliferation and strongly suggest that cooperative DNA binding by these two groups of proteins represent the basis for Hox-induced cellular transformation.

The role of host lymphoid populations in the response of mouse EMT6 tumor to photodynamic therapy.

Photodynamic therapy (PDT) treatment of murine EMT6 mammary sarcoma using Photofrin (10 mg/kg) and light (110 J/cm2) cured all these lesions growing in syngeneic BALB/c mice. In contrast, the same treatment produced initial ablation but no long-term cures of EMT6 tumors growing in either scid or nude mice, the immunodeficient strains sharing the same genetic background with BALB/c mice. No difference was detected in either the level of Photofrin accumulated per g of tumor tissue or the extent of tumor cell killing during the first 24 h after PDT of EMT6 tumors growing in BALB/c or scid mice. The assumption that the difference in tumor cures could be ascribed to the absence of functional lymphoid cells in scid and nude mice was supported by the results of experiments involving the adoptive T-cell transfer into scid mice or bone marrow transfer between BALB/c and scid mice. The adoptive transfer of splenic virgin T lymphocytes from BALB/c mice into scid mice performed 9 days before PDT of EMT6 tumors growing in the recipients was successful in delaying the recurrence of treated tumors. Adoptive transfer done immediately after PDT or 7 days after PDT had no obvious benefit. Even better improvement and a high cure rate of PDT-treated tumors was obtained with scid mice reconstituted with BALB/c bone marrow. In contrast, a marked drop in tumor cure rate was observed with BALB/c mice reconstituted with scid bone marrow. These results suggest that the activity of host lymphoid populations was essential for preventing the recurrence of EMT6 tumors following the PDT treatment used in this study. The contribution of PDT-induced immune reaction may, therefore, be of critical importance for the cure with at least some tumors.

Potentiation of photodynamic therapy-elicited antitumor response by localized treatment with granulocyte-macrophage colony-stimulating factor.

Murine squamous cell carcinoma (SCCVII) cells were genetically engineered to produce marine granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF immunotherapy, based on the peritumoral injection of lethally irradiated GM-CSF-producing SCCVII cells, was examined as adjuvant to photodynamic therapy (PDT) treatment of this tumor. The GM-CSF immunotherapy administered three times in 48-h intervals, starting 2 days before the light treatment, substantially improved the curative effect of Photofrin-mediated PDT. A comparable effect of GM-CSF immunotherapy was observed in the combination with benzoporphyrin derivative-mediated PDT. The tumor-localized GM-CSF immunotherapy alone had no obvious effect on the growth of parental SCCVII tumors. This treatment did not significantly alter the differential peripheral WBC count and appeared not to affect tumor leukocyte infiltration. However, GM-CSF treatment did increase the cytotoxic activity of tumor-associated macrophages against SCCVII tumor cells. It appears, therefore, that tumor-localized immune stimulation by GM-CSF amplifies a PDT-induced antitumor immune reaction, which has a potentiating effect on tumor control.

Photofrin accumulation in malignant and host cell populations of various tumours.

Photofrin accumulation in malignant and host cell populations of various tumours was studied by flow cytometry analysis of cells dissociated from the tumour tissue. The transplantable mouse tumour models included in this analysis were sarcomas EMT6, RIF, KHT and FsaN, Lewis lung carcinoma, SCCVII squamous cell carcinoma (SCC) and slowly growing moderately differentiated AT17 SCC. An example of spontaneous mouse adenocarcinoma was also examined. Staining with specific monoclonal antibodies was used to identify the various cell populations present in these tumours. The main characteristic of Photofrin cellular accumulation was a very high photosensitiser content found exclusively in a subpopulation of tumour-associated macrophages (TAMs). Photosensitiser levels similar to or lower than in malignant cells were observed in the remaining TAMs and other tumour-infiltrating host cells. Photofrin accumulation in malignant cells was not equal in all tumour models, but may have been affected by tumour blood perfusion/vascularisation. Results consistent with the above findings were obtained with SCC of buccal mucosa induced by 9,10-dimethyl-1,2-benzanthracene in Syrian hamsters. The TAM subpopulation that accumulates by far the highest cellular Photofrin levels in tumours is suggested to be responsible for the tumour-localised photosensitiser fluorescence.

Accumulation of benzoporphyrin derivative in malignant and host cell populations of the murine RIF tumor.

The accumulation of benzoporphyrin derivative (BPD) in malignant and host cell populations of the mouse RIF tumor was studied by flow cytometry analysis of cells dissociated from the tumor tissue. The highest cellular BPD levels were found in a subpopulation of tumor associated macrophages (TAMs) characterized by a highly elevated expression of interleukin 2 receptor (F4/80+CD25++), but this selectivity was less pronounced than with photofrin. The BPD levels in tumor associated immune cells other than TAMs were lower than in malignant cells. No significant difference in cellular distribution of BPD was observed with the photosensitizer delivered in liposomes compared to the aqueous solution.

Photofrin accumulation in malignant and host cell populations of a murine fibrosarcoma.

Photofrin (25 mg/kg) was administered to the FsaR fibrosarcoma-bearing mice (either syngeneic or severe combined immunodeficient [SCID]) and the tumors were excised 24 h later. The photosensitizer content in the cells dissociated from tumor tissue was analyzed using flow cytometry. Staining the cell suspensions with the monoclonal antibodies against specific membrane markers served to identify the malignant cells and various types of host immune cells infiltrating the tumor. Photofrin content was also examined in the cells from normal tissues of the tumor-bearing mice (spleen, heart muscle, peritoneal macrophages). The results show a marked heterogeneity in the Photofrin cellular content of FsaR tumor, particularly within the population of tumor-associated macrophages (TAM). The Photofrin levels in some TAM were lower or similar to those in the malignant cells. In contrast, a subpopulation of TAM accumulated very high levels of the photosensitizer, which exceeded by far the levels found in the other tumor cell populations. This TAM fraction was characterized by particularly high expression of interleukin-2 receptors and increased cell size and granularity when compared to the other TAM, which suggests that these macrophages are in the activated state. Their average Photofrin content was almost 13 times higher than in the malignant cells. The lowest photosensitizer levels in the tumor were found in tumor-infiltrating leukocytes other than TAM. In FsaR tumors growing in SCID mice, the pattern of Photofrin distribution in TAM and other cellular populations was similar to that found in tumors growing in syngeneic mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of immune cell infiltration into murine SCCVII tumour by photofrin-based photodynamic therapy.

Cellular populations in the squamous cell carcinoma SCCVII, growing in C3H/HeN mice given Photofrin, were examined at various time intervals during the photodynamic light treatment and up to 8 h later. Cell populations present within excised tumours were identified by monoclonal antibodies directed against cell type-specific membrane markers using a combination of the indirect immunoperoxidase and Wright staining or by flow cytometry. Photofrin-based photodynamic therapy (PDT) induced dramatic changes in the level of different cellular populations contained in the treated tumour. The most pronounced was a rapid increase in the content of neutrophils, which increased 200-fold within 5 min after the initiation of light treatment. This was followed immediately by an increase in the levels of mast cells, while another type of myeloid cells, most likely monocytes, invaded the tumour between 0 and 2 h after PDT. The examination of cytolysis of in vitro cultured SCCVII tumour cells mediated by macrophages harvested from the SCCVII tumour revealed a pronounced increase in the tumoricidal activity of tumour-associated macrophages isolated at 2 h post PDT. It seems, therefore, that the PDT-induced acute inflammatory infiltration of myeloid cells into the treated tumour is associated with functional activation of immune cells.

Enhanced macrophage cytotoxicity against tumor cells treated with photodynamic therapy.

In vitro treatment of human lung adenocarcinoma cells A549 with photofrin-based photodynamic therapy (PDT) resulted in the potentiation of macrophage-mediated killing of these cells assayed either by measuring 3H-thymidine release from the prelabeled target cells, or by determining the survival of A549 cells based on colony formation. The effector cells in these experiments were human promyelocytic leukemia cells HL60 induced to differentiate into macrophages. Very similar results were obtained with the murine squamous carcinoma SCCVII cells treated with PDT and subsequently admixed with mouse peritoneal macrophages. This effect increased with PDT dose and reached its maximum (i.e. complete or nearly complete release of the radioactive label) with the photodynamic treatment that was lethal to 40-50% of cells. In contrast, the PDT treatment of normal mouse kidney cells resulted in only a very limited enhancement of their cytolysis by mouse peritoneal macrophages. The exposure of A549 cells to X-ray irradiation had not affected the macrophage-mediated killing of these cells. The PDT survival curves of A549 cells cultured either alone or with the effector cells showed that the presence of macrophages even at very low effector: target cells ratios enhanced the PDT response of tumor cells. The enhancement ratio of 3.6 (at S = 0.01) was achieved with the effector: target cell ratio 2.5:1, which was the highest ratio tested with this assay. It is suggested that macrophages may recognize potentially repairable damage induced by PDT in tumor cells (presumably lipid fragments exposed in damaged cellular membranes), which helps them to identify the affected cells as their targets.

Cellular levels of photosensitisers in tumours: the role of proximity to the blood supply.

Flow cytometry using the tumour perfusion probe Hoechst 33342 was employed to examine the distribution of photosensitisers in tumour cells located at different distances from the blood supply. Two tumour models, the SCCVII squamous cell carcinoma and FsaR fibrosarcoma growing in C3H/HeN mice, were used in the experiments. Among the photosensitisers tested, only BPD (benzoporphyrin derivative monoacid) exhibited uniform distribution in tumour cells irrespective of their distance from the vasculature. In this respect, 5-aminolaevulinic acid (i.e. its metabolite protoporphyrin IX), di- and tetrasulphonated aluminium phthalocyanines (A1PcS2 and AlPcS4), di- and tetrasulphonated tetraphenylporphines (TPPS2 and TPPS4), Photofrin and bacteriochlorophyll-a (i.e. its metabolite bacteriopheophytin-a) followed BPD in decreasing order in their efficacy of accumulation in tumour cells remote from the blood supply. This photosensitiser property appeared not to depend on tumour type, tumour size, route of photosensitiser administration, time after the administration, photosensitiser lipophilicity or on the presence of host cell infiltrate in the tumour. Following treatment with photodynamic therapy (PDT) in vivo, tumour cells were sorted based on their blood vessel proximity and their survival was determined by colony formation assay. The data demonstrate that the direct killing of tumour cells by Photofrin- and A1PcS2-based PDT decreases with increasing distance of the cells from the blood supply.

Potentiation of photodynamic therapy by immunotherapy: the effect of schizophyllan (SPG).

Treatment of squamous cell carcinoma (SCCVII) bearing mice with the immunostimulant schizophyllan (SPG) raised the relative content of Mac-1 positive host cells infiltrating the tumor and increased photofrin retention in these tumors. In vitro colony formation assay following photofrin-based photodynamic therapy (PDT) in vivo revealed a greater killing of tumor cells in the SPG pre-treated group, particularly pronounced when the tumor excision was delayed for 8 h after PDT. The tumor cure rate increased approximately three times when PDT was preceded by the SPG therapy. In contrast, the administration of SPG after PDT was of no benefit for tumor control.

The effect of differentiation on photosensitizer uptake by HL60 cells.

The capability of human promyelocytic leukemia cells HL60 to be induced to differentiate to various stages along the monocytic or myelocytic pathway was exploited for investigation of the uptake of selected photosensitizers by diverse types of cells of the same origin. The results showed that there was no substantial difference in photofrin uptake between noninduced HL60 cells, immature monocytes, immature neutrophils and cells differentiated along the eosinophilic pathway. In contrast, HL60 cells differentiated into macrophages (HL60 phi) exhibited markedly increased photofrin uptake, which was further enhanced by their pretreatment with bacterial lipopolysaccharide. Similar results were obtained with other photosensitizers tested: di- and tetrasulfonated aluminum phthalocyanines (A1PcS2 and A1PcS4), tetrasulfonated zinc phthalocyanine (ZnPcS4), tetraphenylporphine tetrasulfonate (TPPS4) and benzoporphyrin derivative monoacid (BPD). Despite marked differences in the state of self-aggregation and other chemical properties of these compounds, the degree of their preferential uptake by HL60 phi cells showed very little variation. In a typical experiment, the uptake of these photosensitizers by HL60 phi cells was four to five times higher than the uptake by noninduced HL60 cells. In addition to the fluorometric assay employed in most of the experiments, cellular concentration of A1PcS4 was determined by measurement of elementary aluminum using atomic absorption spectroscopy.

Distribution of Photofrin between tumour cells and tumour associated macrophages.

Photofrin levels in cells derived from SCCVII tumours, excised from mice that previously received the drug, were measured using a fluorescence activated cell sorter (FACS). Concomitantly, in the same cells the FACS was used to measure fluorescein isothiocyanate (FITC) fluorescence that originated from FITC-conjugated antimouse IgG added to the cell suspension before sorting. This later measurement enabled discrimination between IgG negative tumour malignant cells and IgG positive host cells (primarily macrophages). In addition, cellular Photofrin content in 'tumour' and 'host' cells sorted by FACS was determined by chemical extraction. The measurements were performed for the time intervals 1-96 h post Photofrin administration. The data showed consistently higher Photofrin levels in the 'host cells', i.e., tumour associated macrophages (TAM), than in 'tumour' cells. On a per cell basis, at any time point studied there was a minimum of 1.7 times more Photofrin in 'host' than in 'tumour cells', while at 4-12 h postadministration, ratios of up to 3.0 times were observed. This corresponds to ratio values greater than 9, when based on Photofrin content per micrograms cell protein.

Photofrin uptake by murine macrophages.

The uptake of Photofrin by murine peritoneal macrophages in vivo and in vitro was examined. Cellular Photofrin content was measured either by performing a fluorometric assay or by using 14C-labeled drug. For comparison, the uptake of Photofrin by murine SCCVII tumor cells (squamous cell carcinoma) was also examined under the same conditions. The data demonstrate that macrophages have a much greater capacity for Photofrin uptake than SCCVII tumor cells. Photofrin contents at 24 h after drug administration (25 mg/kg) measured 420 +/- 90 (SD), 74 +/- 15, and 15 +/- 2 ng/micrograms of cell protein for peritoneal macrophages, tumor-associated macrophages, and SCCVII tumor cells, respectively. Factors that modify macrophage activity also influence the uptake of the drug by macrophages. The results support the assumption that Photofrin uptake by macrophages is dominated by phagocytosis of highly aggregated components of the drug. In vivo accumulated Photofrin material in peritoneal macrophages, tumor-associated macrophages, and tumor cells has shown very similar in vitro clearance from all three cell types. Only 20-30% of Photofrin was lost from the cells during the initial 24 h, mainly between 1 and 4 h of clearance incubation.

In vitro monocyte maturation in patients with malignant melanoma and colorectal cancer--clinical significance.

In vitro monocyte maturation was studied in 52 malignant melanoma patients, 15 patients with colorectal cancer and 44 healthy donors. Index of maturation (IM) was in malignant melanoma patients 6.3 +/- 5.1%, in colorectal cancer 12.7 +/- 9.6%, and in healthy donors 40.4 +/- 18.0%. The difference between mean values in malignant melanoma patients and patients with colorectal cancer was significant when compared with healthy donors (p less than 0.001). The values in malignant melanoma patients decreased in accordance with stage of the disease and the difference between Stage I and Stage IV was significant (8.2 +/- 4.0% vs. 2.8 +/- 2.0%, p less than 0.01). In patients with colorectal cancer significant difference was established between operated patients and inoperable cases (21.4 +/- 10.0 vs. 5.1 +/- 1.9%, p less than 0.01). Monocyte maturation was influenced by the success of treatment. In patients with complete response the values were significantly higher than in patients with progression (11.7 +/- 6.7% vs. 2.2 +/- 1.5%, p less than 0.001). Autologous serum seems to inhibit the maturation process in vitro in cancer patients, while it apparently has no influence in healthy donors. When a response to treatment was achieved the previously low values of IM increased, while at progression a decrease was noted. The correlation between in vitro monocyte maturation and clinical factors leads to conclusion that the in vitro maturation reflects the in vivo process and may prove useful as a marker of tumor load or spread and may be a sensitive monitor of the treatment effect.