4/21/99

apotosis from necrosis in TEM. I really appeciate it! The replies are

attached below. In addition to the references give, I also found useful

Voume 46 of Methods in Cell Biology; Cell Death, edited by Lawrence .M.

Schwartz and Barbara A. Osborne.

As it turns out, the cells I was looking at are neither apototic nor

necrotic, but now appear to have a strange disorder. But that's another

story...

The replies:

Differentiating apoptosis and necrosis morphologically is based primarily

upon nuclear changes, although there are characteristic cytoplasmic

changes as well. In general (note the wiggle words), necrotic cells swell

and lyse, whereas apoptotic cells shrink and fragment. Chromatin in

apoptotic cells forms electron-dense crescents at the nuclear envelope,

then breaks up.

Apoptotic cells fragment into "apoptotic bodies" that may contain bits of

chromatin. A good place to see characteristic ultrastructural changes of

apoptosis is in lymphoid tissues, where lymphocytes die via apoptosis and

then are phagocytosed by resident macrophages (the ones that are sometimes

called "tingible body macrophages" because of the staining properties of

the apoptotic cell remnants in them.)

Differenting apoptosis from necrosis is a tricky deal. Apoptotic cells

may undergo secondary necrosis, during which they swell and lyse. So,

just because you see necrotic cells doesn't mean that they didn't die

apoptotically. Like everything else, it's complicated; there is a

continuum of change with apoptosis and necrosis at opposite poles and a

lot of stuff in between!

There are lots of good reviews on this topic. The Aug 28, 1998 issue of

Science had a special section on apoptosis, and on page 1302, there is a

series of three electron micrographs of neurons undergoing apoptosis.

One of the first reviews of the subject contains the best collection of

micrographs I've found - "Cell death: the significance of apoptosis" in

the International Review of Cytology 68:251-306, 1980. Another good

review was in Amer J of Pathol 146:3-15, 1995. The title is "Apoptosis,

oncosis, and necrosis: an overview of cell death."

Jane Fagerland

There's lots of stuff on t.e.m. of apoptosis in vertebrate cells (it's

very popular in HIV, cancer, inflammation response etc) and much of it

indicates visible nuclear changes but relative stability of cytoplasm

compared with necrosis.

There was a review article (as a good starting point):

Microscopical Study of Cell Death via Apoptosis by S. Verhaegen

in MIcroscopy and Analysis, January 1998 pp5-7

But you could do a reference or citation 'trawl' on the authors: Kerr,

J.F.; Wyllie, A.H. or Currie

Malcolm Haswell

In response to your question, I did some necrosis versus apoptosis

questions concerning a mycobacterium ulcerans toxin question we had here,

and two papers that were particularly helpful distinguishing the two were

from Scanning Microscopy Vol. 10, No. 1, 1996 pages227-237,by E. Falcieri,

et al, Different Approaches to the study of Apoptosis, and in the same

journal, by Dini et al, pages 239-252, an article entitled Recognition and

Phagocytosis of Apoptotic Cells.

Beth Fischer

Apoptosis: the molecular basis of cell death - Current Communications

in Cell and Molecular Biology 3. L. D. Tomei and F. O. Cope editors.

Cold Spring Harbor Laboratory Press 1991

Cell Death in Biology and Pathology. I.D. Bowen and R.A. Lockshin ed.

Chapman and Hall publs.

John (Keoni) Hardy

The most "conventional" way to detect apoptosis is to look for DNA

fragmentation using in situ hybridization probes. I would not recommend

that you go down that path unless you really need this technique to work

in your lab. There are many in situ probes available to detect apoptotic

cells if you do decide this is what you want.

The DNA fragmentation that occurs during apoptosis will produce "patterns"

in the nuclear chromatin which researchers have used to identify apoptotic

cells.

However, this has the same pitfalls as other morphologic characterizations

(mostly in proving this is what you are looking at).

You might try looking for ways to detect cytoplasmic cytochrome c or

activated

caspases (not easy yet).

Here are some reviews to read:

Baker et al, 1996 Oncogene 12:1-9.

Lincz, 1998 Immuno.and Cell Bio. 76:1-19.

Van Engeland et al, 1998 Cytometry 31:1-9

Solary et al 1998 Cell Bio. and Toxicol 14:121-132.

Green & Reed 1998 Science 281:1309-1312

Dickson 1998 Trends Biotechnol 16:339-342

Cai et al 1998 Biochem. Biophys Acta 1366:151-165.

O'Brian 1998 J. Gen virol 79:1833-1845.

Granville et al 1998 Lab Invest 78:839-913

Kuan and Passaro 1998 Arch Surg 133:773-775.

Paul Webster

Aloha,

Tina

****************************************************************************

* Tina (Weatherby) Carvalho * tina@pbrc.hawaii.edu *

* Biological Electron Microscope Facility * (808) 956-6251 *

* University of Hawaii at Manoa * http://www.pbrc.hawaii.edu/bemf*

tina@pbrc.hawaii.edu