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Cell Growth & Differentiation Vol. 10, 457-465, July 1999
© 1999 American Association for Cancer Research

Retinoblastoma Protein Expression Leads to Reduced Oct-1 DNA Binding Activity and Enhances Interleukin-8 Expression1

Hongquan Zhang, Alexander T. Shepherd, Donna D. Eason, Sheng Wei, Jose I. Diaz, Julie Y. Djeu, Gary D. Wu and George Blanck2

Departments of Pathology and Laboratory Medicine [H. Z., J. I. D.] and Biochemistry and Molecular Biology [A. T. S., D. D. E., G. B.], College of Medicine, and Immunology Program [S. W., J. Y. D., G. B.], H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, Florida 33612, and Department of Internal Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 [G. D. W.]


   Abstract
TOP
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Tumor cell lines with a defective retinoblastoma gene are unable to transcribe the HLA class II genes in response to IFN-{gamma} treatment, and reconstitution of functional Rb rescues IFN-{gamma}-induced class II gene expression. However, the molecular mechanism of Rb rescue of the class II genes is unknown. We have examined the effect of Rb expression on the activation of the promoter for HLA-DRA, the prototype class II gene. Oct-1, a POU domain transcription factor, was identified as a repressor of HLA-DRA promoter activity in the Rb-defective cells. Rb expression led to phosphorylation of Oct-1, thus relieving its repressive effect. Oct-1 has also been shown to repress interleukin 8 promoter activity. Consistent with reduced levels of Oct-1 DNA binding activity in the Rb-transformed cell lines, interleukin 8 expression is higher in these cell lines.


   Introduction
TOP
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
Rb3 , a well-studied tumor suppressor, is required for regulating cell proliferation and differentiation (1 , 2) . Inactivation of the Rb gene due to deletions or mutations has been found in a variety of human tumors, including retinoblastoma, osteosarcoma, and lung, bladder, and prostate carcinomas (3) . Reconstituting functional Rb in some of these tumor cell lines leads to potentially contradictory results. The retinoblastoma cell line Weri-27 and the osteosarcoma cell line Saos-2 have significantly reduced tumor formation capacities after transient transfection of an Rb expression vector and injection into nude mice (4 , 5) . Tumorigenicity is also partially suppressed by stable expression of Rb in the Rb-defective bladder carcinoma cell line 5637, the Weri-27 cell line, and the Saos-2 cell line (6) . In some of these studies, Rb expression in the Rb-defective tumor cell lines had no effect on the growth rate or morphology of the in vitro cultured tumor cells (6 , 7) . Discrepancies between the growth characteristics of the Rb-transformed cells in vitro and the effectiveness of Rb expression in eliminating tumor growth in vivo may reflect a host’s defense mechanism cooperating with Rb in the prevention of tumorigenesis.

Whereas an extensive body of work on Rb has focused on its growth suppression function, there is emerging evidence indicating that Rb plays a role in the modulation of the immune functions of tumor cells (8, 9, 10, 11, 12, 13 , 17) . The aberrant production of IL-6 by neoplastic cells is regarded as a strong contributory factor in the growth of a variety of malignancies, especially multiple myeloma, leukemia, and lymphomas (11 , 12) . A survey of the Rb status in the cells representing acute myeloblastic leukemia indicated that Rb defects in the acute myeloblastic leukemia blast cells closely correlate with the increased IL-6 mRNA expression in these cells. Also, a reduction of Rb expression with antisense oligonucleotide in the normal blast cells induces IL-6 mRNA expression, and overexpression of Rb represses an IL-6 promoter reporter construct (8 , 13) .

The HLA class II genes encode heterodimeric cell surface proteins that present antigens to CD4+ T helper cells. The interaction between the HLA class II molecules on tumor cells and the T-cell receptors may help the host’s immune system to recognize tumor cell antigens and to mount an immune response against tumor cells (14 , 15) . Studies from our laboratory have established that Rb is required for the IFN-{gamma} inducibility of HLA class II expression in tumor cell lines. Tumor cell lines defective for Rb expression express little or no HLA class II after IFN-{gamma} treatment; however, HLA class II inducibility can be restored by exogenous expression of Rb (9 , 10 , 17) .

In this study, we used a combination of EMSAs and transfection experiments to examine the effect of Rb expression on the HLA-DRA promoter. Oct-1, a ubiquitously expressed POU domain transcription factor, was determined to play a role in the effect of Rb expression on the activity of the promoter of the HLA-DRA gene, which encodes the heavy chain of the HLA class II DR molecule. This work also led to the conclusion that Rb enhances IL-8 expression by an Oct-1-related mechanism.


   Results
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 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
Oct-1 Binding Activity to the HLA-DRA Promoter Is Reduced in Rb-transformed Cell Lines.
To elucidate the mechanism of Rb rescue of HLA-DRA inducibility, we compared HLA-DRA promoter protein DNA binding activities in the Rb-defective and Rb-transformed human tumor cell lines (Table 1Citation ; Refs. 6 , 7 , 9 , 16 , and 17 ). Nuclear extracts were obtained from 5637 cells and its subclones, as well as MDA-468-S4 (S4) and its Rb-transformed subclone S4-MTRb-1. Equal amounts of these extracts were analyzed by EMSA, and the oligonucleotide probes used in the experiment included the previously characterized HLA-DRA promoter X, Y, and octamer box elements (18) . Oct-1 binding activity was markedly reduced in the Rb-transformed subclones compared to the Rb-defective control subclones (Fig. 1ACitation , compare Lane 2 with Lane 1 and Lane 4 with Lane 3). Protein binding to the HLA-DRA Y box is shown in Fig. 1BCitation . The identity of Oct-1 was verified by anti-Oct-1 antibody supershift (Fig. 1ACitation , Lanes 6–8) and by binding competition with unlabeled wild-type or mutated octamer element (Fig. 1ACitation , Lanes 9–12). Nonspecific binding to the octamer element was completely eliminated by an excess of both wild-type and mutated octamer oligonucleotides (Fig. 1ACitation , lower bands).


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  		Table 1 Cell lines used in this study

 


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  		Fig. 1. Reduced Oct-1 binding to the HLA-DRA promoter octamer element using extracts from Rb-transformed cells. Equal amounts (5 µg) of nuclear extracts from the Rb-defective and Rb-transformed cells (Table 1)Citation were analyzed by EMSA. A 26-bp synthetic oligonucleotide including the HLA-DRA promoter octamer element was used as a probe. A, cells lines are as indicated (Table 1Citation ; Lanes 1–4). The identity of the Oct-1-DNA complex was verified by antibody supershift (Lanes 6–8) and binding competition using an excess of unlabeled wild-type and octamer-mutated oligonucleotides (Lanes 9–12) using nuclear extract from the 1A4 cells. B, DNA-protein complexes formed using extracts from 1A4 and 12-27 cells and the HLA-DRA Y box as probe are shown in the top panel. Oct-1 DNA binding activity for the same extracts is shown in the bottom panel. C, cells were either left untreated (Lanes 1–3; 5637, 1A4, and 12-27, respectively) or treated with 400 units/ml IFN-{gamma} (Lanes 4–6; 5637, 1A4, and 12-27, respectively). The upper arrow indicates the Oct-1-DNA complex. The lower arrow indicates a nonspecific protein-DNA complex (NS)

 
Because the HLA-DRA promoter is activated by IFN-{gamma} treatment, we examined whether the difference in Oct-1 binding in the Rb-defective versus the Rb-transformed cells is maintained in the presence of IFN-{gamma}. EMSA was performed using nuclear extracts from IFN-{gamma}-treated and untreated 5637 cells and its subclones, 1A4 and 12-27 (Table 1)Citation . The result indicated that IFN-{gamma} treatment caused a slight decrease in the Oct-1 DNA binding activity in the Rb-defective 5637 subclone 1A4 as well as in the Rb-transformed subclone 12-27 (Fig. 1CCitation , compare Lanes 1–3 with Lanes 4–6). However, the difference in Oct-1 binding between the Rb-defective and Rb-transformed cells remained unchanged in the presence of IFN-{gamma}. IFN-{gamma}-treated Rb-defective cells retained a higher Oct-1 binding activity than their Rb-transformed counterparts.

The Repressive Effect of Oct-1 on HLA-DRA Promoter Activity Is Relieved by Rb Expression.
To determine whether the difference in the Oct-1 DNA binding activities could have an effect on HLA-DRA promoter activity, we devised a strategy to assess the impact of endogenous Oct-1 DNA binding activity on an HLA-DRA promoter reporter construct. We generated two promoter reporter constructs by subcloning the -176 to +45 region of the HLA-DRA promoter (19) into pGL3 luciferase vector. One construct has the wild-type octamer element of the HLA-DRA promoter (pDRAlucWT), and the other construct contains the mutant octamer element generated by substituting four nucleotides within the octamer box (pDRAlucOCTmut; "Materials and Methods"). The octamer element has been shown to have a repressive effect on HLA-DRA promoter activity, and the nucleotide substitutions eliminate this repressive effect (19) . These substitutions also represent the mutated octamer sequence used in the EMSA described above (Fig. 1A)Citation . We reasoned that if the higher level of Oct-1 DNA binding activity in the Rb-defective cells affected HLA-DRA promoter activity, then the mutations in the octamer element would have a greater effect in the Rb-defective cells than in the Rb-transformed cells. That is, if the endogenous Oct-1 differences between the two cell types could affect promoter activity, then the mutations in the octamer box should lead to a greater relief of repression in the Rb-defective cells. The wild-type and mutant constructs were transiently transfected into the Rb-defective 5637 cells and the Rb-transformed subclone 12-27. Transfected cells were treated with IFN-{gamma}, and the cell lysates were assayed for luciferase activity by scintillation counting. In the Rb-defective 5637 cells, the activity of the HLA-DRA promoter with the mutated octamer box was increased by 1.7-fold compared with that of the wild-type promoter (Fig. 2)Citation , consistent with the original data indicating that Oct-1 is a negative regulator of HLA-DRA promoter (19 , 20) . However, in the Rb-transformed 12-27 cells, there was no significant difference in promoter activity between the wild-type and mutant promoter constructs (Fig. 2)Citation , consistent with reduced Oct-1 binding in this Rb-transformed cell line.



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  		Fig. 2. Repressive effect of Oct-1 on HLA-DRA promoter activity relieved by Rb expression. The wild-type (pDRAlucWT) or octamer-mutated HLA-DRA (pDRAlucOCTmut) promoter luciferase constructs were transiently transfected into Rb-defective 5637 cells or the Rb-transformed subclone, 12-27. The cell lysates were assayed for luciferase activity by scintillation counting. In 5637 cells, the mutant construct had a 1.7-fold increase in promoter activity compared with that of the wild-type construct. In 12-27 cells, there was no significant difference in promoter activity between the wild-type and mutant constructs.

 
Increased IL-8 Secretion in Rb-transformed Cells.
Oct-1 is a transcriptional repressor for the human IL-8 gene promoter (21) . We wanted to determine whether Rb expression also relieves the repressive effect of Oct-1 on the IL-8 promoter. First, we tested whether reconstitution of Rb in the Rb-defective 5637 cells affects the amount of IL-8 secretion. Culture media was collected 24 h after cell plating and analyzed by ELISA for IL-8, which revealed that IL-8 was secreted by the Rb-defective and Rb-transformed 5637 cells, and that the secretion of IL-8 was increased in the Rb-transformed subclones (Fig. 3)Citation . After 24 h of culture, the amount of IL-8 secretion from the Rb-transformed subclone 12-27 is 1.6-fold higher than that for 1A4 cells, which were paired with 12-27 cells as the Rb-defective control for this experiment. The amount of IL-8 secretion from the Rb-transformed cell line 10-1 was 5.3-fold higher than that of the Rb-defective 1A1 subclones, paired with 10-1 as a control in a separate experiment.



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  		Fig. 3. IL-8 secretion by the Rb-defective and Rb-transformed 5637 subclones. Each 5637 subclone was plated in triplicate, and culture media were collected at 24 h after plating. The concentration of IL-8 in the culture media was determined by ELISA, and data represent the mean ± SE of triplicate samples.

 
The Repressive Effect of Oct-1 on IL-8 Promoter Activity Is Relieved by Rb Expression.
Next, we wanted to determine whether increased IL-8 secretion by the Rb-transformed cells could be attributed to a lower level of Oct-1 DNA binding activity in these cells. We first used a strategy similar to the strategy used in the analysis of the effect of the Rb-related Oct-1 differences on the HLA-DRA promoter, as described above. As described above, this strategy was designed to determine the effect of the differences in the endogenous levels of Oct-1 DNA binding activity on IL-8 promoter activity. IL-8 promoter luciferase constructs containing a wild-type or mutated octamer element (21) were transfected into the Rb-defective and Rb-transformed 5637 cells, and cell extracts were assayed for luciferase activity. In the parental 5637 cells, the construct with the mutated octamer element had a 2.5-fold higher promoter activity compared with the wild-type construct, whereas in the Rb-transformed subclone 12-27, there was a 1.5-fold increase in promoter activity for the construct with the mutated octamer element (Fig. 4)Citation . Northern blotting was performed to be certain that the increased level of IL-8 secretion by the Rb-transformed cells reflected an increase in IL-8 mRNA levels. The mRNA bands were quantified and normalized to 28S rRNA. Setting the ratio of IL-8 mRNA:28S rRNA in parental 5637 cells as 1, we observed relative ratios of 1.73 and 4.23 in the Rb-defective 1A4 subclone and the Rb-transformed 12-27 subclone, respectively (Table 2)Citation .



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  		Fig. 4. Repressive effect of Oct-1 on HLA-DRA promoter activity relieved by Rb expression. The wild-type or octamer-mutated IL-8 promoter luciferase constructs were transiently transfected into the Rb-defective 5637 cells or the Rb-transformed subclone, 12-27. The luciferase activities were determined as described in the legend to Fig. 2Citation . In 5637 cells, the octamer-mutated IL-8 promoter construct had a 2.5-fold increased promoter activity compared with the wild-type construct. In 12-27 cells, the mutant construct had a 1.5-fold increase in promoter activity compared with the wild-type construct.

 

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  		Table 2 IL-8 mRNA in the Rb-defective and Rb-transformed cellsa

 
Oct-1 Binding to the IL-8 Promoter Octamer Element Is Reduced in Rb-transformed Cells.
As indicated above, Oct-1 binding to the HLA-DRA promoter octamer element is reduced in Rb-transformed cells. One report (22) has indicated that different kinase activities leading to Oct-1 phosphorylation might differentially affect the binding of Oct-1 to different octamer elements. To verify that Oct-1/IL-8 octamer box binding was similarly reduced in the Rb-transformed cells, an EMSA was performed using nuclear extracts from the parental 5637 cell line and its subclones 1A4 and 12-27 (Table 1)Citation . A 20-bp double-stranded oligonucleotide including the human IL-8 gene promoter octamer element was used as a probe. Oct-1 binding activity was detected for 5637 and 1A4 cells but was not detectable for the Rb-transformed cell line 12-27 (Fig. 5Citation , Lane 4). Binding competition with the unlabeled wild-type or mutated octamer probe and Oct-1 antibody supershift confirmed the specificity of the Oct-1/DNA complex (Fig. 5Citation , Lanes 5–8).



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  		Fig. 5. Oct-1 binding to the IL-8 promoter octamer element. EMSA was performed as described in the legend to Fig. 1Citation , and the probe used in this experiment is a 20-bp synthetic oligonucleotide including the IL-8 promoter octamer element. Oct-1 DNA binding activity can be detected in extracts from the Rb-defective parental 5637 cell line and the 1A4 subclone (Lanes 2 and 3), and Oct-1 DNA binding activity is nearly absent in extracts from the Rb-transformed 12-27 cells (Lane 4). The Oct-1-DNA complex was verified by binding competition (Lanes 5 and 6) and antibody supershift (Lanes 7 and 8). The large complex below Oct-1 binds both the octamer element and the mutated octamer element, indicating that this complex is nonspecific. However, the complex is apparently disrupted by the anti-Oct-1 antibody, for unknown reasons.

 
Culture Media from the Rb-transformed Cells Enhances Neutrophil Migration.
IL-8 is a potent chemotactic factor for neutrophils and lymphocytes. It may also be involved in the angiogenesis and metastasis of some tumors (23 , 24) . To assess a possible functional significance of the differences in IL-8 secretion among the 5637 subclones, we used a microwell chemotaxis chamber assay. In this experiment, the chemotactic agent (i.e., culture media from the cell lines) and neutrophils were separated by a polycarbonate filter and incubated at 37°C for 1 h. The filter, which trapped migrated neutrophils, was then washed and stained. Migrated neutrophils on the filter were microscopically evaluated and counted (Fig. 6Citation ; "Materials and Methods"). The number of migrated neutrophils was increased by culture media from the Rb-transformed 12-27 cells (Fig. 6Citation , compare D with C). Table 3Citation shows the neutrophil migration rates from two independent experiments. In one experiment, a 1:1 dilution of the Rb-transformed 12-27 culture media led to a migration rate of 30% versus 2% for a 1:1 dilution of the Rb-defective 1A4 culture media. Additional experiments indicated similar differences in the migration rates generated by culture media from Rb-transformed versus Rb-defective cell lines.



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  		Fig. 6. Enhanced neutrophil migration in response to culture media from Rb-transformed cells. Neutrophil migration was evaluated by the microwell chemotaxis chamber assay ("Materials and Methods"). Photomicrographs (magnification, x40) show representative fields of the migrated neutrophils in response to (A) RPMI 1640 with 10% FCS (negative control), (B) 100 ng/ml recombinant human IL-8 (positive control), (C) culture media from the Rb-defective 1A4 subclone, or (D) culture media from the Rb-transformed 12-27 subclone. Arrows indicate the neutrophils trapped by the filter.

 

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  		Table 3 Neutrophil migration in response to culture media from the Rb-defective and Rb-transformed cellsa

 
Hyperphosphorylation of Oct-1 in Rb-transformed Tumor Cells.
To elucidate the mechanism of reduced Oct-1 binding to the HLA-DRA and IL-8 octamer elements (Figs. 1ACitation and 5Citation ), we performed Western blotting to evaluate Oct-1 protein levels in the nuclear extracts from both the Rb-defective and Rb-transformed 5637 subclones. Equivalent amounts of Oct-1 were detected in the Rb-defective 1A4 and 1A1 subclones and the Rb-transformed 12-27 and 10-1 subclones (Fig. 7A)Citation , excluding the possibility that differences in Oct-1 protein levels accounted for promoter binding differences in these cells.



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  		Fig. 7. Hyperphosphorylation of Oct-1 in the Rb-transformed cells. A, Western blotting ("Materials and Methods") of Oct-1 in the Rb-defective and Rb-transformed 5637 subclones (Table 1)Citation . Nuclear extract (30 µg) from each 5637 subclone was analyzed. B, hyperphosphorylation of Oct-1 in 12-27 cells. 5637 subclones were metabolically labeled by [32P]orthophosphate. Oct-1 was immunoprecipitated and analyzed by SDS-PAGE followed by autoradiography. Phosphorylated Oct-1 is indicated in the top panel by the arrow. This same gel was transferred and blotted by anti-Oct-1 antibody (arrow, bottom panel).

 
Because phosphorylation of Oct-1 reduces its DNA binding activity (22 , 25) , we analyzed the phosphorylation state of Oct-1 in the 5637 subclones. Cultured cells were metabolically labeled by [32P]orthophosphate, Oct-1 was immunoprecipitated from nuclear extracts by anti-Oct-1 monoclonal antibody, and the immunoprecipitates were analyzed by SDS-PAGE followed by autoradiography ("Materials and Methods"). Results revealed that Oct-1 was phosphorylated in both the Rb-defective and Rb-transformed 5637 subclones (Fig. 7BCitation , top panel), but that phosphorylation of Oct-1 was increased in the Rb-transformed 12-27 subclone at 24 h and 48 h after plating of the cells.

This same gel was then transferred and blotted with anti-Oct-1 antibody (Fig. 7BCitation , bottom panel) to determine the total amount of Oct-1. Oct-1 phosphorylation levels in the Rb-defective and Rb-transformed 5637 subclones were quantified and normalized to total Oct-1 as detected by the Western analysis ("Materials and Methods"). Setting the Oct-1 phosphorylation levels (phosphorylated Oct-1:total Oct-1) in the Rb-defective 1A4 subclone at 24 h after plating as 1, we obtained a relative level of 1.7 in the Rb-transformed 12-27 subclone cultured during the same time period. The differences in Oct-1 phosphorylation between the 1A4 and 12-27 cells remained constant at 48 h after plating (Table 4)Citation .


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  		Table 4 Quantitative analysis of Oct-1 phosphorylation

 

   Discussion
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 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 References
 
Relief of Oct-1-mediated Repression by Rb.
The ubiquitously expressed transcription factor Oct-1 functions as either a positive or a negative regulator of a wide range of cellular genes, including genes for snRNA (26) , H2B (27) , immunoglobulin (28) , CD11 (29) , CD20 (30) , IL-2, and IL-4 (31) . The regulation of these genes is dependent on Oct-1 interaction with the octamer motifs (ATTTGCAT or its variants) present in the promoters or enhancers (27 , 28 , 32) . The genes regulated by Oct-1 encode products participating in a variety of cellular processes, ranging from housekeeping to cell growth and differentiation (27 , 28 , 32) .

As discussed above, IFN-{gamma} treatment of Rb-defective cells does not lead to HLA class II gene transcription or leads to only a very low level of transcription. Rb expression rescues HLA class II inducibility (9 , 10 , 17) . Data described in this report indicate that the repressive effect of Oct-1 on the HLA-DRA promoter is alleviated by reconstitution of functional Rb (Figs. 1Citation and 2Citation ). However, relief of Oct-1-mediated repression is unlikely to represent an explanation for the entire effect of Rb on the HLA class II genes because HLA-DRB, which encodes the other polypeptide of the HLA-DR molecule, does not have an octamer site in its promoter. Also, no octamer sites have been described for the HLA-DP genes, the induction of which is also dependent on Rb. However, it is interesting to note that the HLA-DRA gene seems more heavily dependent on Rb than do other HLA class II genes (9) , suggesting the possibility that Oct-1-mediated repression and a second Rb-related mechanism simultaneously regulate HLA-DRA induction.

Oct-1 is also a transcriptional repressor of the IL-8 promoter, in which a variant of the octamer element overlaps a CCAAT-enhancer-binding protein binding site (21) . As with the HLA-DRA promoter, the repressive effect of Oct-1 on the IL-8 promoter is relieved by expression of Rb (Fig. 4)Citation , with the expected consequence of higher levels of IL-8 production by Rb-transformed cells. This is the first report that the IL-8 gene expression is regulated by Rb.

Phosphorylation of Oct-1 by Rb Expression.
The Western blotting and immunoprecipitation analyses of Oct-1 (Fig. 7)Citation revealed that Oct-1 is hyperphosphorylated in Rb-transformed cells, which explains the reduced Oct-1 binding to the HLA-DRA and IL-8 promoters (Figs. 1Citation and 5Citation ). Other than Rb expression, only one process has been reported to alter Oct-1 DNA binding activity or phosphorylation: mitosis (25 , 33) . In mitosis, Oct-1 becomes highly phosphorylated, with a concomitant loss of DNA binding activity (25) . Although phosphorylation of Oct-1 by either protein kinase A or protein kinase C (22) in vitro reduces its binding to the octamer element, the Oct-1 kinase(s) that functions in vivo is unknown (33) .

Oct-1 and Growth Control.
Oct-1 is a transactivator involved in the cell cycle regulation of H2B gene transcription and in the activation of the snRNA genes, which are presumably important for rapid cell division. Re-expression of Rb in the Rb-defective tumor cells probably leads to reduced Oct-1 binding to the H2B and snRNA promoters, facilitating Rb-mediated G1 growth arrest. If so, this would be the first E2F-independent link between Rb and growth control. However, this proposal is not supported by the conclusion that the vast majority of H2B gene regulation occurs at the level of H2B mRNA stability, not at the level of transcription (34) . Thus, Oct-1 is thought to effect only a minor increase in H2B promoter activity. However, it is not known whether Oct-1 is required for basal level H2B or snRNA transcription. Resolution of this issue awaits a direct test of the hypothesis that Oct-1 DNA binding activity enhances cell growth rates.

Antitumor Immunity and Rb.
Baskar et al. (35 , 36) have found that tumor cells transfected with and expressing class II molecules can immunize mice against a subsequent challenge of parental, class II-negative tumor cells. Similar conclusions have been drawn using a rat model system (37) . Thus, tumor cells defective for Rb, which cannot express HLA class II after exposure to IFN-{gamma}, may be less immunogenic.

IL-8 secretion now represents a second immune function regulated by Rb. Enhanced neutrophil migration in response to culture media from the Rb-transformed cells is consistent with this increased IL-8 secretion having a functional impact on the neutrophils, although it is not known whether there could be additional, Rb-dependent components of the culture media enhancing neutrophil migration. A role for IL-8 in antitumor immunity is suggested by the following: (a) recombinant IL-8 inhibits the proliferation of some non-small cell lung carcinoma cell lines, and anti-IL-8 antibody eliminates this effect (38) ; (b) Taxol, an antitumor drug, induces the secretion of IL-8 but not IL-1 or IL-6 from ovarian carcinoma cell lines (39) ; (c) differentiation-inducing agents, such as retinoic acid, calcitriol, and sodium butyrate, up-regulate IL-8 expression in HL-60 cells (40) ; and (d) tumor-infiltrating lymphocytes express high levels of IL-8 (41) . However, there are also reports contradicting the proposal that IL-8 enhances antitumor immunity (24 , 42 , 43) . Melanoma cell lines with higher IL-8 secretion have a stronger tendency for metastasis in nude mice (43) , and the ability of IL-8 to induce angiogenesis in tumors is regarded as the contributing factor in tumor development (42) . Although a determination the exact roles of HLA class II and IL-8 in the antitumor response requires additional investigation, it is clear that Rb can regulate at least two tumor cell immune functions in addition to cell growth and differentiation.


   Materials and Methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
References
 
Cell Culture.
The human bladder carcinoma cell line 5637 (ATCC HTB-9; Refs. 16 and 17 ) was cultured in RPMI 1640 supplemented with 10% FCS (Hyclone), 100 units/ml penicillin-streptomycin, 3 mM L-glutamine, and 1 mM sodium pyruvate. 1A4 and 1A1 are Rb-defective 5637 subclones transformed with the G418-resistant gene. 12-27 and 10-1 are 5637 subclones transformed with an Rb expression vector and the G418-resistant gene (Table 1Citation ; Refs. 6 and 16 ). They are maintained by adding 200 µg/ml Geneticin (Life Technologies, Inc.) to the culture media used for culturing the 5637 cells. S4-MTRb-1 is a Rb-transformed subclone of the human breast carcinoma cell line S4 (Table 1Citation ; Refs. 7 and 9 ). S4 and S4-MTRb-1 were grown in DMEM:F12 (Mediatech), 10% calf serum (Hyclone), 100 units/ml penicillin-streptomycin, 3 mM L-glutamine, and 50 ng/ml epidermal growth factor (Boehringer Mannheim).

Preparation of Nuclear Extracts and EMSAs.
Nuclear extracts were prepared as described by Schreiber et al. (44) , with minor modifications. Briefly, cells (1 x 106 cells/10-cm dish) were washed twice with cold PBS and once with hypotonic buffer containing 20 mM HEPES, 1 mM EDTA, 1 mM EGTA, 20 mM sodium fluoride, 1 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM DTT, and 0.5 mM phenylmethylsulfonyl fluoride. Cells were then lysed in 300 µl of hypotonic buffer containing 0.2% NP40 and protease inhibitors (0.5 µg/ml leupeptin, 2 µg/ml aprotinin, and 50 µg/ml antipain; Boehringer Mannheim). Nuclei were separated from cytosolic components by centrifugation at 15,000 x g for 30 s and resuspended in 100 µl of high salt buffer (20 mM HEPES, 840 mM sodium chloride, 1 mM EDTA, 1 mM EGTA, 40% glycerol, 1 mM sodium fluoride, 1 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, and the protease inhibitors). After 30 min of intermittent mixing at 4°C, the tubes were centrifuged at 15,000 x g for 5 min (4°C). The supernatants (nuclear extracts) were aliquoted and stored at -70°C for later use. Protein concentration was determined by the BCA protein assay following the manufacturer’s protocol (Pierce).

The probes used for the EMSAs have been described previously (19 , 21 , 45) . The sequences of the synthesized wild-type and mutant HLA-DRA octamer oligonucleotides, with the octamer element underlined and the mutated nucleotides in lowercase letters, are as follows: wild-type, 5'-AGAGTAATTGATTTGCATTTTAATGG-3' (sense strand) and 5'-CCATTAAAATGCAAATCAATTACT-3' (antisense strand); and mutant, 5'-AGAGTAATTGccaTGgATTTTAATGG-3' (sense strand) and 5'-CCATTAAAATcCAtggCAATTACT-3' (antisense strand). The HLA-DRA Y box oligonucleotide sequences are 5'-AAATATTTTTCTGATTGGCCAAAGAGTAAT-3' (sense strand) and 5'-ATTACTCTTTGGCCAATCAGAAAAAT-3' (antisense strand). The sequences of the oligonucleotides used for the IL-8 promoter octamer probes are as follows: wild-type, 5'-TCATCAGTTGCAAATCGTGG-3' (sense strand) and 5'-TCCACGATTTGCAACTGATG-3' (antisense strand); and mutant, 5'-TCATCttgTGCAAtgCGTGG-3' (sense strand) and 5'-TCCACGcaTTGCAcaaGATG-3' (antisense strand). EMSAs were performed according to Yu et al. (46) . Nuclear extract (5 µg) was incubated with 0.025 pmol of [{alpha}-32P]dCTP or [{alpha}-32P]dATP end-labeled double-stranded probe for 30 min at 30°C in a 20-µl incubation mixture containing 1 µg of poly(deoxyinosinic-deoxycytidylic acid) (Sigma) and 5 µg of bovine serum albumin (Boehinger Mannheim). For competition or supershift experiment, 5 µg of nuclear extract were preincubated with a 10-fold or 100-fold molar excess of unlabeled double-stranded oligonucleotides or 1 µl of monoclonal antibody (anti-Oct-1 and the control anti-epidermal growth factor receptor were both bought from Upstate Biotechnology) for 30 min at room temperature and then combined with the labeled probes. The protein-DNA complexes formed were separated on an 8% nondenaturing polyacylamide gel and autographed.

Reporter Constructs, Transfections, and Luciferase Assays.
To generate the wild-type HLA-DRA luciferase reporter (pDRAlucWT), the -176 to +45 fragment of HLA-DRA promoter was excised from pDRACAT (19) and subcloned into pUC18. The fragment was then excised from pUC18 using Asp718 and SalI. This fragment was then cloned into the Asp718 and XhoI sites upstream of the luciferase reporter gene in the pGL3 vector (Promega). The corresponding mutant construct (pDRAlucOCTmut) was generated by subcloning the same DRA promoter fragment with four nucleotide substitutions within octamer box (5'-ATTTGCAT-3' to 5'-ccaTGgAT-3') into the pGL3 vector. Both the wild-type and octamer mutant DRACAT plasmids were generous gifts of Dr. Laurie Glimcher (Harvard University School of Public Health, Boston, MA). Both DRA luciferase constructs were verified by DNA sequencing. The wild-type and octamer-mutated IL-8 promoter luciferase reporter constructs have been described previously (21) .

For transient transfections, 5637 or 12-27 cells were plated at a density of 2 x 105 cells/35-mm dish and incubated at 37°C with 7.5% CO2. Each transfection was performed five times. After 24 h, 100 ng (DRA promoter reporters) or 1 µg (IL-8 promoter reporters) of DNA was transfected using a liposome method (Trans IT-LT1; Mirus Corp.) following the manufacturer’s recommendations. Six h after transfection, cells were washed once with PBS, received fresh media, and were cultured for an additional 42 h. Cells were then harvested and lysed in 100 µl of 0.25 M Tris-HCI (pH 8.0) by three rounds of freezing (95% ethanol/dry ice) and thawing (37°C). The whole cell lysates were centrifuged at 15,000 x g for 5 min at 4°C. The supernatants were assayed for luciferase activity using commercial reagents (Promega). Light emissions were measured by a liquid scintillation counter (Beckman).

Isolation of the Cytoplasmic RNA and Northern Blotting Analysis.
Cytoplasmic RNAs from the cultured 5637 cells and its subclones were extracted and stored as an ethanol precipitate as described previously (47) . Northern blotting was performed as described by Luca et al. (24) , with minor modifications. RNA (30 µg) from each sample was fractionated in a 1% denaturing formaldehyde/agarose gel. The fractionated RNA was then transferred to a nylon filter membrane (Hybond-N; Amersham) and UV cross-linked (UV-Stratalinker 1800; Stratagene). Prehybridization was performed at 42°C in 6 x SSC containing 50% deionized formamide, 0.2% SDS, and 100 µg/ml salmon sperm DNA. A 0.5-kb EcoRI fragment representing human IL-8 cDNA (23 , 24) was purified from the agarose gel by using the Qiaquick kit (Qiagen) and labeled with [{alpha}-32P]dCTP using a nick translation kit (Sigma). Hybridization was carried out overnight at 42°C. The membrane was washed twice with 2 x SSC and 0.1% SDS for 30 min at 50°C and then washed once with 0.1 x SSC and 0.1% SDS for 30 min at 60°C and exposed to Kodak film with an intensifying screen at -70°C. Another set of RNA samples was fractionated by electrophoresis under the same conditions described above and stained with ethidium bromide to evaluate the integrity and concentration of the ribosomal RNAs.

Quantitation of the IL-8 mRNA and the corresponding 28S rRNA bands was done using ImageQuaNT computer software (version 4.2a, Molecular Dynamics).

ELISA and Neutrophil Migration Assay.
Culture media for 5637 subclones were collected at 24 h after plating. ELISA determination of IL-8 concentrations was performed in the Cytokine Core Laboratory at University of Maryland at Baltimore (Baltimore, MD). Culture media from triplate samples of each subclone were analyzed. Neutrophil migration assay was performed by using a 48-well microchemotaxis chamber (Neuro Probe, Inc., Cabin John, MD) following the manufacturer’s protocol. Neutrophils were isolated from the peripheral blood of normal adult volunteers (South West Florida Blood Bank, Tampa, FL; Ref. 48 ).

Immunoprecipitation and Western Blotting.
5637 subclones (50–80% confluent monolayer in 10-cm dish) were cultured in 3 ml of labeling media [phosphate free-DMEM (Life Technologies, Inc.) containing 5% dialyzed FCS and 0.1 mCi/ml inorganic phosphate (NEX054; DuPont New England Nuclear)]. Nuclear extracts were isolated from the labeled cells as described above. For immunoprecipitation of Oct-1, 5 µl of mouse antihuman Oct-1 antibody (Upstate Biotechnology) and 10 µl recombinant protein G agarose (Life Technologies, Inc.) were added to 250 µl (100 µg) of nuclear extracts and incubated for 2 h at 4°C. Immunoprecipitates were recovered by centrifugation at 2,500 rpm for 5 min and washed three times with PBS at 4°C. Immunoprecipitates were analyzed by SDS-PAGE using a 10% gel, the wet gel was exposed to Kodak film for the detection of 32P-labeled Oct-1, and the band intensities were quantified using ImmageQuaNT software (Molecular Dynamics). To immunoblot Oct-1, proteins were transferred to a polyvinylidene difluoride membrane (DuPont New England Nuclear Research Products) in a buffer containing 20 mM Tris, 192 mM glycine, and 20% methanol overnight at 4°C. The membrane was then blocked in 10 ml of PBS containing 5% milk and 0.1% Tween 20 overnight at 4°C. Antibody blotting was performed by incubating the membrane in 10 ml of 5% milk containing 10 µl of anti-Oct-1 (1:1,000 dilution) for 2 h at room temperature. The membrane was washed three times with PBS containing 0.1% Tween 20 for 5 min each time at room temperature, and 2.5 µl (1:12,500 dilution) of horseradish peroxidase-labeled rabbit antimouse antibody (Life Technologies, Inc.) were added and incubated with the membrane for 1 h. The reaction was detected using enhanced chemiluminescence reagents (Amersham).


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by American Cancer Society Grant RPG-98-184-01-CIM, American Heart Association of Florida Grant 9601461, an American Lung Association of Florida grant (to G. B.); and NIH Grant AI39368 (to G. D. W.). Back

2 To whom requests for reprints should be addressed, at 12901 Bruce B. Downs Boulevard MDC 7, Tampa, FL 33612. Phone: (813) 974-9585; Fax: (813) 974-7280; E-mail: gblanck{at}com1.med.usf.edu Back

3 The abbreviations used are: Rb, retinoblastoma protein; IFN-{gamma}, interferon-{gamma}; IL, interleukin; EMSA, electrophoretic mobility shift assay; H2B, histone 2B; snRNA, small nuclear RNA. Back

Received for publication 4/ 2/99. Revision received 5/14/99. Accepted for publication 5/18/99.


   References
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 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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