The HL-60 cell line has been used as an in vitro model system for studying cellular and molecular mechanisms involved in the proliferation and differentiation of normal and leukemic cells of the hematopoietic, or white blood cell lineage. This cell line, derived from a patient with a type of leukemia, is an immortal cell line with distinct myeloid characteristics. As such, HL-60 cells may be grown in suspension culture for extended periods and may be induced to differentiate to a number of different cell types. These characteristics are similar to stem cell lines. In the hierarchy of stem cells, the HL-60 cells are multipotent as opposed to totipotent, or pluripotent (Figure 1)

• Totipotent: able to form every type of cell in the body

• Pluripotent: able to form most cell types, but not limited to a specific system

• Multipotent: able to form multiple cell types, but restricted to a specific lineage/system

Experimental studies with HL-60 cells have examined 1) control mechanisms of normal white blood cell differentiation, 2) potential therapeutic approaches for patients with leukemia, 3) possible mechanisms of rapid regulation of gene transcription, and 4) specific cellular oncogene expression in relationship to particular hematopoietic differentiative lineages. Additionally, HL-60 cells provide a readily available source of mRNA for cloning of important granulocyte and monocyte enzymes, and cytokines. Cytokines are small proteins secreted by the cell which bind to cell receptors to induce differentiation, proliferation, cell survival, apoptosis, or a variety of other effects.

Various chemical agents have been used to induce differentiation via signal transduction (Figure 2) in HL-60 cells into lymphocytes, monocytes, and granulocytes (consisting of basophils, neutorphils, and eosinophils). The cell type of HL-60 cells in culture is predominantly promyelocytes, which spontaneously differentiate to morphologically mature cells including myelocytes, metamyelocytes, and neutrophils. The addition of specific inducing agents accelerates this differentiation with most cells acquiring morphological, functional, enzymatic, and surface membrane antigen characteristics of mature granulocytes (Figure 3). Most of these cells are involved in human immune system function.

In this laboratory exercise, we will be examining HL-60 cell response to chemical agents known to induce differentiation, specifically dimethylsulfoxide (DMSO) and phorbol 12-myristate 13-acetate (PMA). DMSO induces differentiation of HL-60 cells into a cell population dominated by granulocytes. PMA treatment of HL-60 cells increases the proportion of monocytes in the cell population. (Figure 4).

The central dogma of molecular biology is the principle of directional flow of genetic information from DNA to RNA to protein. The flow of information involves DNA replication, transcription (copying information without “language” change) from DNA to RNA, and translation (language change from nucleotide sequence to amino acid sequence) from RNA to protein. Genetic expression of the eukaryotic cell is reflected in the products produced, i.e., RNA or proteins. Control of this genetic expression is exerted at different levels during this process: 1) genomic, 2) during transcription, 3) during RNA processing, 4) during translation, and 5) post translationally

DNA is transcribed into mRNA which is translated into Protein.

Here's a few links to important videos:

Transcription

Translation

When HL-60 cells are induced to differentiate, they are no longer immortal and subsequently will eventually undergo a process known as apoptosis. Apoptosis is programmed cell death and differs from the other two types of cell death, autophagy and necrosis. Autophagy is self-ingestion of the cell and may occur when apoptosis is blocked. When a cell undergoes necrosis, it swells, bursts, and decomposes. In this course we will examine the differences in apoptosis and necrosis, focusing on the mechanisms involved in apoptosis.

1. Necrosis is the result of an injury to the cell, while apoptosis is a naturally occurring programmed cell death.

2. During the process of necrosis, the cell becomes swollen until it bursts into cellular fragments. Apoptosis, however, begins with nuclear fragmentation followed by the formation of cellular fragments known as apoptotic bodies. These fragments are then engulfed by phagocytosis.

3. When a cell responds to an injury and undergoes necrosis, the cell debris induces an inflammatory response. There is no inflammatory response during apoptosis.

Necrosis vs. Apoptosis Video

As we will be focusing on cellular differentiation, it is necessary that we have a clearer understanding of the apoptotic processes. Generally, apoptosis takes place in four steps:

General Apoptotic Pathways (takes place in the cytoplasm):

1. Adapter proteins bind with two identical initiator caspases to form an active enzyme

2. The initiator caspases cleave and activate the executioner caspases.

3. The executioner caspases cleave hundreds of substrates resulting in cellular apoptosis, fragmenting the cell into small apoptotic bodies.

4. Phagocytosis of apoptotic bodies.

There are two types of apoptotic pathways, extrinsic and intrinsic. When cells are treated with PMA or DMSO, apoptosis takes place via the extrinsic pathway. However, induced differentiation may also occur with other agents via an intrinsic pathway. Examples of each of these and the steps involved are as follows:

Extrinsic apoptosis (involves transmembrane receptor-mediated interactions)

The Death Receptor Pathway

1. Death ligands attach to

2. Death receptors or integrin receptors (cell surface) attracting

3. Adapter proteins form death-inducing complex (DISC) resulting in

4. Caspase activation: Initiator caspase (8) – Executioner caspase (3, 6, 7)

5. Substrate cleavage follows and leads to

6. Cell death

Intrinsic apoptosis

The Mitochondrial Pathway is the main mode used in vertebrates and is in response to cytotoxic stress.

1. Apoptotic stimuli of

2. Family of BCL-2 proteins (located in the cytoplasm) cause

3. mitochondrial outer membrane permeabilization (MOMP)

4. cytochrome c released and promotes

5. adapter protein oligomerization

6. apoptosome complex forms and promotes

7. Caspase activation: Initiator caspase (9) – Executioner caspase (3)

8. substrate cleavage forming apoptotic bodies

9. cell death and phagocytosis

Green, D. R. (2011). Means to an End Apoptosis and Other Cell Death Mechanisms. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

Lodish, H. et al. (2013). Pp., 1006-1016. Molecular Cell Biology, 7^th ed. W. H. Freeman and Company, New York, New York.

Plopper, G. (2013). Pp.437-445. Principles of Cell Biology. Jones & Bartlett Learning, Burlington, Massachusetts.

Hofman, W. (2009). Cell and Molecular Biology of Nuclear Actin. International Review of Cell and Molecular Biology, Vol 273. DOI: 10.1016/S1937-6448(08)01906-6.

Xu, Y., et al. (2010). Nuclear translocation of β-actin is involved in transcriptional regulation during macrophage differentiation of HL-60 cells. Molecular Biology of the Cell, Vol 21: 811-820.

Discussion of the Xie paper: “Fibronectin-mediated Cell Adhesion Is Required for Induction of 92-kDa Type IV Collagenase/Gelatinase (MMP-9) Gene Expression during Macrophage Differentiation” by Bei Xie et al. (1998) J. Biol. Chem. 273 (19), 11576-11582.

Learning objectives:

1. Can you identify the hypothesis, experimental design and conclusions? Briefly describe these sections

2. Are the conclusions of this article supported by the data? Do they answer the original question?

3. Locate and name the author(s), the date the article was published, and the journal the article was published in.

4. Now, try to find both a primary article and a review article that is related to this topic. What kinds of differences can you see between the two types of article?

5. Define any terms you are not familiar with.

Macrophages degrade the extracellular matrix (ECM) either:

1. directly by secreting MMPs and their specific inhibitors, or

2. indirectly by releasing cytokines such as interleukin-1(IL-1) and tumor necrosis factor-α (TNF-α) which then stimulate fibroblasts or synovial cells to secrete MMPs.

MMP-9

• 92 kDa type IV collagenase/gelatinase

• Breaks down collagen, gelatin, fibronectin (FN), and elastin in the ECM

• Major MMP produced by human macrophages

• Necessary for extravasation, migration, and tissue remodeling following inflammatory response

• Macrophage MMP-9 production is closely associated with cellular differentiation

• Differentiation into macrophages results in increased cell adhesion and spreading on ECM

• FN is key in promoting cell adhesion and other functions associated with monocytes/macrophages

· promote migration and phagocytosis of these cells

· modulation of cytokine expression (i.e., IL-1, TNF-α, and macrophage-colony stimulating factor (M-CSF))

· mediated by two surface receptors, α5β1 and α4β1 integrins found on monocytes/macrophages

· α5β1 integrin recognizes the cell-binding domain of FN at the RGDS protein sequence (arginyl-glycyl-aspartyl-serine)

· α4β1 integrin acts as the receptor for the CS-1 region of FN

• PMA (phorbol 12-myristate 13-acetate) promotes differentiation into macrophages in human peripheral blood, as well as in HL-60 cells

• It is essential that protein kinase C (PKC) is activated for differentiation to progress

• Gene expression of PKC-β in HL-60 cells is necessary for macrophage differentiation to take place

• HL-525 cells (a PMA-resistant HL-60 variant) lose PMA resistance when treated with trans-retinoic acid (trans-RA) which enhances PKC-β expression.

• PKC-β expression is restored in HL-525 cells by PKC-β gene transfection

Hypothesis: The critical steps involved in the induction of MMP-9 gene expression during macrophage differentiation in HL-60 cells treated with PMA and human peripheral blood monocytes treated with either PMA or M-CSF are:

1. FN-mediated cell adhesion and spreading

2. Mediated mainly through α5β1 integrin signaling

3. PKC-β is essential for the production of MMP-9

1. Central dogma of molecular biology: events are ordered from transcription to translation, so that DNA produces RNA, which produces protein

2. Control of this genetic expression: Occurs at genomic level, during transcription, during RNA processing, during translation, and during posttranslational modifications.

3. Differentiation: formation of a more specialized cell from a less specialized cell type

4. Dimethylsulfoxide: colorless liquid which can dissolve both polar and nonpolar compounds, and on its own can serve as a signal to induce certain cells to differentiate

5. Genetic expression: production of functional RNA and/or proteins

6. Hematopoietic: pertaining to the formation, development, and differentiation of blood cells

7. HL-60 cell line: human acute promyelocytic leukemia cells growing indefinitely in culture which can be studied as a model system for white blood cell differentiation

8. Human fibronectin: extracellular matrix glycoprotein that binds to integrins at the cell surface to promote adhesion of cells to collagens

9. Immortal cell line: cells growing in culture that can continue to divide indefinitely

10. In vitro model system: an artificial environment outside of the living organism

11. Integrin receptors: transmembrane proteins that mediate attachment of a cell to the tissues around it and can also act as a cellular receptor

12. Multipotent: able to form multiple cell types, but restricted to a specific lineage/system

13. Phorbol 12-myristate 13-acetate: activates enzyme protein kinase C, can act as a potent tumor promoter, and induces differentiation of human leukemia cells

14. Pluripotent: able to form most cell types, but not limited to a specific system

15. Signal transduction: intermolecular reactions involved in transferring and amplifying extracellular signals detected by the cell

16. Totipotent: able to form every type of cell in the body

17. Transcription: DNA sequence is copied into mRNA

18. Translation: mRNA directs the formation of a specific protein at a ribosome in the cytoplasm